McTips

McTips = McNamara tips. 

For several years I organized by year (links below) but now (2024) simpler to add content (mostly near the top, occasionally add to bottom) here. 

Tips & Procedures (web page)  http://confocal.jhu.edu/mctips/tipsprocedures .

** 20240123U note: I am currently 20% time at Ross Image Center, so new McTips will be rare, and new 2024 page not worth organizing. I put an antibody solutions centrifugation (cold minifuge rotor) tip near the top of this page (just past the web links).

20240809F Leica SP8 HyD detectors linear range up to 60 photons per second (each) - see http://confocal.jhu.edu/mctips/leica-sp8-hyds-linear-range-to-60-photons-per-microsecond

2021 PC Tips for microscope and analysis workstations http://confocal.jhu.edu/mctips/pc_tips_2021

2022 PC Tips for microscope and analysis workstations http://confocal.jhu.edu/mctips/pc_tips_2022

2022_part2 PC Tips (started 20220705U) http://confocal.jhu.edu/mctips/pc_tips_2022_part2

Confocal Sweetest Spots (web page) http://confocal.jhu.edu/mctips/confocalsweetestspot  

  Online Image Data Repositories - blog (20210427W web page)  image-data-repositories

    MPMicro "Multi-Probe Microscopy" (1500 pages - please do not print out) https://works.bepress.com/gmcnamara/2/ 

Multiplex fluorescence microscopy (web)  http://confocal.jhu.edu/mctips/multiplex ... in addition to BV's could add some CPNs, StarBrights, NovaFluors, SuperBrights, Spark's, Alexa Fluor and/or Aluora TSA, etc.

     Alexa Fluor 610-X performance calculations wrt FISHscope http://confocal.jhu.edu/mctips/af610-x   (gm note: SulfoRhodamine 101 = SR101, Brightness=125 aboutthe same as AF610)

     StreamBio UK LinkBright CPNs labeling kits available (1/2003) from Sigma-Aldrich (hopefully low shipping and total costs) http://confocal.jhu.edu/mctips/streambiouk-linkbright-cpn-kits

McTips 2024 http://confocal.jhu.edu/mctips/mctips-2024 (no PDF) note: -2024 vs _2023 for next URL)

McTips 2023 http://confocal.jhu.edu/mctips/mctips_2023 (no PDF)

McTips 2020 PDF go to: McTips 2020 download at https://works.bepress.com/gmcnamara/90

McTips 2019 PDF go to: McTips 2019 download at https://works.bepress.com/gmcnamara/85

McTips 2018 PDF go to: McTips 2018 download at https://works.bepress.com/gmcnamara/84

McTips 2017 PDF go to: McTips 2017  download at https://works.bepress.com/gmcnamara/81/

McTips 2017 Direct download is    https://works.bepress.com/gmcnamara/81/download 

Factoids (started 01/2022) http://confocal.jhu.edu/mctips/factoids

FISHscope Quick Tips (startup, close out session)  http://confocal.jhu.edu/mctips/fishscope-quick-tips -- FISHscope main page is   http://confocal.jhu.edu/current-equipment/fishscope 

* Quick Leica SP8 confocal microscope tip (see more info on our SP8 page from current equipment):

I recommend (we have Leica SP8 Klondike linear scanner - most SP8's are the same sub-model):

HyD detectors, Photon counting mode ... not max linear counting rate is 6 Mcps (6 million counts per second) = 6 counts per microsecond ( 6 counts/usec) or 0.6 counts per 100 nanoseconds.

          At very high counts the safety interlock should trigger, resulting in all values ZERO until the user stops scanning (i.e. 1000x1000 x 100 planes Z-series could be lots of zeros).

             "About 10 counts" is sufficient to recognize a nicely labeled DAPI nucleus;

              "about 100 counts" is good but not great immunofluorescence (depends on background!),

              "about 300 counts" is very nice signal (hence my always instructing users to acquire 16-bit mode on SP8,

              "over 1000 counts" may be a waste of time -- unless your experiment needs very high dynamic range. 

600 Hz line scan rate (enables full range of zoom

10 Line accumulation as a starting point, more or less depending on user needs. Max is 16 line accumulation (Leica SP8, FV3000RS would be 1 line "average"). I note that with tyramide signal amplification (TSA), 10x to 100x brtighter than standard secondary antibodies (see table later on this page), "1 may be enough" (2 or more would make a prettier image but may or not matter for p-values). 

1 Frame accumulation as starting value, more if needed. 

Modest laser power, usually 0.5% or 1% for 405nm laser, 1 or 2 percent for any of 488, 552, 638nm lasers, depending on what fluorophores are used (ex. DAPI, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 647).

Sequential scan tracks, usually ... can combine blue (DAPI) and NIR (AF647Plus) in moany experiments (3plex plus DAPI), such as: (i) AF555Plus, (ii) AF488Plus, (iii) DAPI + AF647Plus. 

Always have detection bandpass AT LEAST 10nm away from any laser line - even if the laser line is not used in that wscan track. If no NIR, turn off the 638nm laser.

 

20240916M:  #1.5H Coverglasses High Precision best for high resolution confocal microscopy etc

#1.5H Coverglasses High Precision best  for high resolution confocal microscopy etc

20240916M:

For best image quality on confocal microscopes, high resolution fluorescence microscopy - ex: single molecule RNA FISH on FISHscope - you should image through 170 um thick coverglass made of borosilicate glass (ibidi 180 um cyclic olefin polymer is also ok - should be consistent thickness even if "off by 10 um"). Coverglasses are numbered as:

#00 ... ~70um ... see https://analyticalscience.wiley.com/content/article-do/importance-coverslips-microscopy 

#0 ... ~100 um

#1 ... ~130 um

#1.5 ... ~170 um

#1.5H ... 170 um +/- 5 um H = High Precision

#2 ... ~210 um

#3 ... ~300 um

#4 ... ~400 um

#5 ... 500-600 um

See   https://analyticalscience.wiley.com/content/article-do/importance-coverslips-microscopy  for ranges of the other specifications.

Note: . hemacytometers use thick coverglass to prioritize rigidity for correct volume and almost always would image on a low magnfication, low or modest numerical aperture objective lens).

I strongly recommend you / your lab to buy #1.5H coverglasses and #1.5H (aka "0.170")  coverglass imaging dishes, so that ALL your experiments are compatible with high resolution microscopy. Also, segregate existing #0, #0 etc and label your collection so no one in the lab uses the wrong coverglass. 

I note that #00 would add ~100 um working distance, and #0 would add ~70um working distance, for those experiments where you need to prioritize working distance. I also note that -- in principle -- GPU enabled spatial deconvolution could correct for aberrations induced by imaging through non-optimal coverglass.

Lastly: occasionally TWO coveglasses get stuck together. Careful inspection can detect these - you do not want your critical slide to be un-imageable (at high resolution) due to 2 coverglasses. How to detect? (1) put hold two standard coverglasses together and you will likely see Newton's rings (interference fringes) - same may occur if two are stuck together, (2) you may be able to see and/or feel a coverglass is "thicker than normal" - but you need to pay close attention.

Marienfeld (Germany) is the main manufacturer of #1.5H - they make many formats and list them on their web site

https://www.marienfeld-superior.com/precision-cover-glasses-thickness-no-1-5h-tol-5-m.html

There are many U.S. distributors, such as Zeiss online store, ThorLabs.

For imaging coverglasses

* Mattek  p35g-0-170-14-c     (apparently only 14 mm diameter imaging area)

https://www.mattek.com/store-category/35mm-dishes/page/2/

https://www.mattek.com/store/p35g-0-170-14-c

$350 for case of 75, so $4.67 per dish.

ibidi 

https://ibidi.com/35-mm-dishes/176-375--dish-35-mm-high-glass-bottom.html#/29-surface_modification-15h_170_%C2%B5m_5_%C2%B5m_d_263_m_schott_glass_sterilized/171-pcs_box-400_individually_packed

current (9/2024) prices:

 $355 for   60 = $5.92   each. ... 10% off if buy 5 or more ... so if 

buy 300 then $5.325 each.

$2010 for 400 = $5.025 each. ... 10% off if buy 5 or more ... so if buy

2000 then $4.5225 each ($9045 total) ... maybe GCRF store could do that (the image cores I manage could not scale this).

WPIinc.com - I did not see #1.5H (170um +/- 5um) high precision dishes on their web site.

Mattek standard 14 mm are $1.90 each (case of 200 for $380, 9/2024 price)

https://www.mattek.com/store/p35g-1-5-14-c-case/

so you may want to buy both "1.5" and "0.170" cases, with labels reminding users "routine" vs "high resolution" imaging projects. 

***

Borosilicate glass should always be used (coverglass and slide) - but you cannot assume!!!

9/2024 I was reading Lab Manager magazine and its co-wrapped sibling Clinical Lab magazine, and was shocked to read that some pathology labs (i.e. clinical diagnoses) MIGHT use the wrong glass. See

https://www.clinicallab.com/does-cover-glass-matter-in-digital-pathology-27982

for the story. I think the emphasis should have been to use #1.5H coverglasses (the ~2x higher cost is trivial compared to the under $0.01 per slide). I was organizing my prsentation (9/2024) for the BioTrac 2024 Spatial Biology conference, and had put in a slide  on the need for the right coverglass (#1.5H = 170 um), so then added the above link and to not assume you are bring sold the right glass.

 

 

20240916M: New Optical Clearing methods preprint - image deeper into organoids, brain slices etc

20240916M: New Optical Clearing methods preprint - image deeper into organoids, brain slices etc

Note: text emphasizes “BSA #1” (cells in culture: better proliferation, less toxicity) – implying some BSA products (and potentially different lots of same product) are better, worse, much worse. For example, different Sigma-Aldrich products have different prices (cheaper in Kg plus quantity – awkward if not a good product for your cells, organoids, larvae etc), and different purity (some lack IgG, but pricier).

Inagaki 20240915 bioRxiv - SeeDB-Live Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo Imai.pdf

https://www.biorxiv.org/content/10.1101/2024.09.13.612584v1

12 videos and one suppl table

https://www.biorxiv.org/content/10.1101/2024.09.13.612584v1.supplementary-material

***

Ed Boyden (brain slices etc):

In Vivo Optical Clearing of Mammalian Brain

https://www.biorxiv.org/content/10.1101/2024.09.05.611421v2

https://www.biorxiv.org/content/10.1101/2024.09.05.611421v2.full.pdf

https://www.biorxiv.org/content/10.1101/2024.09.05.611421v2.supplementary-material

Note: I am a big fan of Ed Boyden and their lab. Ed will win a Nobel Prize (maybe 10/2024!) for Optogenetics (with Karl Deisseroth, maybe one more researcher), hopefully some mention of Expansion Microscopy (ExM).

 

 

Ca++ ion experiments of FV3000RS

20240801H - pasted from an email to a new user whose lab has a lot of experience on the FV3000RS confocal microscope.

Ca++ imaging expt advice – from long experience: start with cells with a simple positive control – and this might take more than one imaging dish (helps if your lab mates tell you the folder/file of one of their recent successful experiments, so we can load their FV3000RS settings). Classic control is Ionomycin (A23187) in Ca++ containing medium. For cell the right receptor, ATP, Carbachol (Ach analog), or some other “high positive control” ligand is ok (many GPCR’s signal predominantly through IP3 second messenger to activate Ca++ ion responses). Some researchers go the extra step of: (1) start timelapse (say 1 minute at 30 fps), (2) ATP or similar physiological ligand and let Ca++ signal subside (1-5 minutes), then (3) ionomycin+Ca++ ions.

In the early days of Ca++ imaging, researchers also included an EDTA (or EGTA?) chelator step, to measure “F0” (lowest fluorescence). This has mostly gone out of fashion when using “intensitometric” Ca++ indivators (GCaMP# fluorescent protein biosensor, Fluo-3, Fluo-4, etc), vs early research ratiometric indicators such as Fura-2 (340/380nm excitation ratio, ~510nm emission, widefield microscopes since nearly all confocal microscopes lack those UV wavelength, and 340nm is phototoxic).

FYI - Fluo-3AM, Fluo-4AM, or (in the old days) Fura-2AM esters (or similar indicators loaded with "AM": easy to overload the cells. This can cause the Ca++ indicator to become a Ca++ chelator, that suppresses Ca++ ion response; The AM ester gets cleaved to result in the ion sensitive indicator + formaldehyde, which means high concentration of the AM ester could result in "fixation" of your cells. Rare(?) but can happen: some cell types (or cells from unusual organisms than typical human HeLa experiments) migt not have sufficient cytoplasmic "non specific esterases" to cleave the AM ester - this would result in the added reagent simply equilibrating in the cytoplasm, resulting in signals that are not Ca++ responsive. The reagent can also end up in non-cytosolic compartment(s), such as lysosomes, other endosomes, possibly ER - or extruded from the cytoplasm (usually through an MDR 'drug resistance' transporter), resulting in usually unwanted or no signal.

 

 

Brilliant and SuperBright Blocking Buffers for BV421 etc

20240730U

Brilliant and SuperBright Blocking Buffers for BV421 etc

* I am a big fan of BV421 and related "polymer fluorophores". Won Nobel Prize in Chemistry in 2000 (electron conducting polymers). 

BV421 is much brighter than standard small dye fluorophores, E.c. 2.500,00 M-1cm-1, QY 0.6, Brightness = Ec*QY/1000 = 1500 vs Fluorescein (pH 8.0) 81 and Alexa Fluor 488 similar B.

* one mystery is why two or more Brilliants tend tio bind each other and/or "non-specifically" bind cells, calibration beads, etc (and why single product is not a problem). 

https://www.colibri-cytometry.com/post/blocking-brilliant-dye-interactions

introduction -- see web page for many more details, lots of flow cytometry scatterplots:

The issue

Interactions between Brilliant dyes on antibody conjugates. Creates compensation-like artefacts where cells staining with one Brilliant-conjugated antibody are artificially positive for others.

 

The reagent

The appropriately named Brilliant Stain buffer.

Available from BD and ThermoFisher:

https://www.thermofisher.com/order/catalog/product/00-4409-42

https://www.thermofisher.com/order/catalog/product/SB-4401-42?SID=srch-srp-SB-4401-42

https://www.bdbiosciences.com/en-gb/products/reagents/flow-cytometry-reagents/research-reagents/buffers-and-supporting-reagents-ruo/brilliant-stain-buffer.566349

Also available in a more concentrated format:

Brilliant Stain Buffer Plus (bdbiosciences.com)

 

What is it?

Probably monomers of the base SIRIGEN dye, according to Florian Mair. The spectrum of the BS buffer resembles that of Super Bright 436, as we'll see later.

 

Alternatives

None, just the variants above. Using less antibody limits the artefacts.

 

What it’s supposed to do

Reduce dye-mediated interactions between Brilliant Violet, Brilliant Ultraviolet, Brilliant Blue and Super Bright conjugated antibodies.

 

Brilliant Stain Buffer also helps reduce background in human whole blood assays because it contains polyethylene glycol (PEG). A lot of fluorophores contain PEG, so the presence of PEG in the buffer swamps interactions between serum antibodies and the fluorophores. Why do people have anti-PEG antibodies? Well, there's PEG in vaccines, too, and a lot of people got vaccinated recently. Read more here.

 

 

 

 

Alexa Fluor Plus secondary antibodies 3x brighter 1.3x more expensive ... Tyramide Signal Amplification (TSA) even brighter somewhat pricier

20240627Thur - summary of sections below on recent reagents for (hopefully) improve your immunofluorescence microscopy

Ultra quick summary: "Brighter is better" ... if you have specific labeling.

GM's rule(s) of thumb for tissue sections: if red blood cell autofluorescence is brighter than your specific antibody labeling

1. buy new reagents (I am assuming you had me help you configure the microscope settings during training for your experiment). You are wasting your time, your money, and microscope time doing experiments with one of more mediocre or bad reagents.

2. identifying red blood cells (RBCs) in your tissue section:

(i) adult human and mouse RBCs lack DAPI nuclear labeling since they lack nuclei (if your DAPI labeling is bad, you need to fix this too);

(ii) human RBCs are ~5 um diameter, nearly all nucleated human cells are >7 um diameter (though in tissue sections, not all cuts are through the equator of a round cell or long acis of a flat cell.

(iii) RBCs autofluorescence is typically "approximately equal" in the green and red channels (i.e. Alexa Fluor  488 for green, Alexa Fluor 555 or Alexa Fluor 568 for red), wheras single fluorescent antigen labeling will be (hopefully) bright in one channel, same as rest of tissue background in the other channel.

(iv) your choice of routine controls is up to you and your lab - that said, single stained controls of "antigen rich" tissue sections is highly prudent -- and you can and probably should include representative image data in your supplemental files (i.e. PDF suppl. text and some raw data). For example, most CD markers (CD3, CD4, CD8, CD20, CD68) will be present in lymph node and spleen tissue sections ('red pulp' of spleen will also have a massive number of RBCs and partially digested RBCs).

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  * 20240701M see nice linkedin thread on topic - both PipeBio's URL and the comments, https://www.linkedin.com/posts/pipebio_recombinant-antibodies-are-less-than-5-of-ugcPost-7213534716845563905-fwuJ 

Product names are not meant as endorsements. Acronyms ("VHH") are explained in "the literature" (peer-reviewed, preprints, product pages, press releases). 

Assumption: standard primary antibody from rabbit or mouse. One hour of confocal microscope time ($30/hr) using standard secondary antibody.

************

Quick Summary of best options (Nov 2014 - GM's opinion) - table below has mre options, more details.

Standard 2ndary antibody:                                                     $1.00 per slide, 1x brightness ... i.e. 1:100 dilution of 1 mg/mL from tube

Recombinant Alexa Fluor PLUS 2ndary antibody:                  $1.30 per slide, 3x - 7x brightness ... no more dead donkeys, goats etc

Tyramide signal amplification (TSA) (HRP-polymer-2ndAb): $6.50 to $10 per slide, 10x to 100x  brightness ... and may need less 1st Ab.

==> I am a fan of direct labeled antibodies, ex: BV421 anti-CD8, brightness depends on how much antigen is present, whether you need to do antigen retrieval if using FFPE tissue sections, whether the microscope is configured correctly, exposure time (widefield) or confocal settings, more.

IBEX Community https://ibexcommunity.com -- nice web site focused on multiplex immunostaining - also known as Spatial Biology - many tips and validated reagents online, open community access (reminder: validated antibodies need to be stored correctly in your laboratory). In pubmed search for: Radtke A IBEX.

One nice IBEX tip for research experiments - from Radtke 2022 Nat Protoc (https://pubmed.ncbi.nlm.nih.gov/35022622 ) is that fixation with 1% paraformaldehyde fixes tissue (i.e. mouse tissues, human cells in culture) nicely but most antibodies do not require antigen retrieval. If you are "translating" your experiments to pathology lab long term stored human FFPE tissue, this "1% solution" will not help you a lot, but if you are asking research questions with specimens for which you control the fixative (ex. mouse tissue, human 3D organoids, human cells in culture) you may want to evaluate the 1% PFA approach (also incubation time with the fixative and wash and storage).

**********

Reminder:

"old antibodies die, please throw them away." 

My play on:  D. Macarthur; "Old soldiers never die, they just fade away". (Douglas died).

You are responsible for making sure all your reagents are working well. If you take old tube(s) of antibody out of the back of the refrigerator, you should verify they all work (and re-store them in a way so they keep working). I stongly recommend diluting each in appropriate buffer and using a cold rotor in a mini-centrifuge mini-fuge, minifuge) to "spin down" aggregates into pellets, and then carefully aspirate-for-use the supernatants. Just using "dilution out of tube" is likely to spray aggregates all over your specimen. 

It only takes on person in your lab to leave antibodies out on a bench overnight or over weekend to ruin them. 

A useful analogy for old antibodies: if you put chicken eggs in your refrigerator at home in January 2020 -- the start of the covid-19 pandemic -- and then found them today, would you eat them? I hope not (be sure to have health insurance before doing so - especially if consume raw). 

*********

Brighter options imply proportionally greater signal (and signal to noise ration) and/or decrease in acquisition time (lower cost per specimen field of view). Not all possibilities are permuted in the table. I used Fab below (fragment antigen binding; VH_CH1 + VL-CL; historically made by specific protease digestion of IgG ad then purify the Fab's from the Fc fragment crystallizable domain), but scFv (single chain fragment variable, VH-linker-VL or sometimes VL-linker-VH) is single chain equivalent, so simpler for recombinant protein production.

Not in table below: DNA-PAINT (single molecule localization Ab-oligo + fluorophore-oligo, antibody-PAINT (especially Fast Kd Ab-PAINT, scFv-PAINT, nanobody-PAINT), hybridization chain reaction (HCR, detecting either a DNA sequence, RNA sequence, Ab-oligo, protein-protein interaction etc). I am a big fan of many of these. 

Also not in the table: the mounting medium you use matters. Elsewhere in this web site I discuss D2O instead of H2O (Maillard 2020 Chem Sci; also Alexa Fluor 610-X AF610-X, brightest of 42 fluorophores they tested; potentially deuterated glycerol, though expensive compared to D2O) to bosst intensity (photons out) of many read and near infrared dyes; some users still use VectaShield with DAPI: (i) VectraShield quenches many CyDyes (i.e. Cy3, Cy5, also some Alexa Fluor trademark named dyes are CyDyes) maybe not a good idea, (ii) often with DAPI in the VectraShield, which is just a way to badly decrease contrast [you can apply DAPI with the secondary antibodies; if using direct label antibodies, can apply with them ... or switch to a NIR DNA dye, perhaps DRAQ5 or To-Pro-3, apply with antibodies); some users use ProlongGold - a product around longer than some of the users have been alive: consider Prolong Diamond or Prolong Glass ... and read the instructions: need to cure at least 24 hours in open space to vent volatile compound(s) that keep them liquid. 

Ideal future in my (George McNamara) opinion: $1 per slide very bright modern fluorophore(s) all recombinant direct label Fab or scFv or primary nanobodies with custom C-terminal tail, simple and efficient labeling of BV421 (Brightness = 1500 vs Alexa fluor 488 or EGFP Brightness ~60; Brightness = Extinction coefficient * Quantum Yield / 1000, for BV421 2.5M * 0.6 / 1000). Using Fab (~50 kDa)  instead of intact IgG (~$155 kDa) eliminates binding to FcR and FcRn receptors (good to avoid); decreases number of lysine (for NHS ester) or cysteines (for maleimide); for 1 ug protein, monomeric Fab has ~1.5x more antigen binding sites than IgG (two antigen binding sites per IgG); custom C-terminal tail: (i) antigen binding site is near N-terminal end so best to avoid, (ii) tail can be whatever length and sequence is desired (optimal for brightness vs total cost [and for companies, profit ... caveat intellectual property protection and rights). 

Some options for immunofluorescence microscopy detection
Option Brightness

Reagent Price (est)

[1st or 1st + detection reagents)

Comment

Nano-Tag or Chromotek VHH nanobody 2ndary Ab

ex: Alpaca anti-rabbit  AF488 VHH

    1/3x         $1 + $1

As formatted from Pleiner et al 2018 JCB (PubMed 29263082; Ewers comment 29444803) (licensed to both Nano-Tag.com and Chromotek (now ptglab.com see Nanobody page) dimmer; but enables multiplexing of multiple mouse monoclonal antibodies etc.

Hypothesis: nanobodies with optimized tail (academic lab with protein synthesis and labeling capability; ThermoFisher etc if they get their act together) whatever the maker wants (ex. 1x, 3x, 5x ...)

       $1 + $1

(or multiplier for the detection reagent)

GM hypothesis/suggestion: Pleiner 2018 suppl table has the sequences of all the VHH used --> instead of "default C-terminal tail" in Pleiner (20+ years of nanobodies) a "custom tail' with optimized conjugation sites for NHS ester (lysine), maleimide (cysteine), non-canonical amino acid(s) (ncAA; click chemistry) could enable optimized brightness, photochemistry (no cross quenching of the fluorophores; optional FRET to diversify colors; FLIM etc).

Further refinement: BV421-streptavidin to mAb-biotin, etc.

Direct labeled Fab - classic dye, ex. Alexa Fluor 488-anti-CD4

ex: mouse (recombinant) Fab fragment monoclonal antibody (~50 kDa) with degree of labeling (DoL) 1-2 Alexa Fluor 488 fluorophores.

    1/9x (?)           $0.33

Direct labeled antibody (IgG) - classic dye, ex. Alexa Fluor 488-anti-CD4

GM note: this could be much brighter as Fab-(custom tail) as hypothesized above for 2ndary VHH nanobody. Could further optimize with BV421 (one or multiple) or BV-tandem (multiplex) on custom tail.

Direct labeled antibody (IgG) - classic dye, ex. Alexa Fluor 488-anti-CD4

ex: mouse monoclonal antibody (~155 kDa) with degree of labeling (DoL) 4 Alexa Fluor 488 fluorophores.

    1/3x           $1 Common in flow cytometry; rare in fluorescence microscopy
Direct labeled antibody - modern fluorophore, ex. BV421-anti-CD4)      3x           $1 Common in flow cytometry; rare in fluorescence microscopy. BV### also available (ex. BV570)

Standard 2ndary antibody (ex. goat-anti-mouse; donkey-anti-rabbit)

     1x        $1 + $1 Standard of care since ~1965 (the fluorophores, microscopes, detectors have improved a lot over ~60 years)

Recombinant 2ndary antibody (ex. goat-anti-mouse; donkey-anti-rabbit)

 

     1x       $1 + $1

==> no more sacrificing small and/or large furry animals for your experiments; more consistent reagents.

Pricing (usually) identical to standard from dead animals 2ndary antibodies - likely more profitable since no animal feed, more consistent products. 

"Plus" fluorophore 2ndary Ab      3x      $1 + $1.30 See below for thermoFisher Alexa Fluor Plus secondary antibodies - Plus recombinant secondary antibodies also available.
"Plus" Recombinant 2ndary antibody (ex. goat-anti-mouse; donkey-anti-rabbit)      3x      $1 + $1.30

* Plus = High brightness.

* Recombinant = high consistency, no more dead furry animals.

https://www.thermofisher.com/antibody/secondary/query/Superclonal%20plus 

Tyramide signal amplification (TSA)

ThermoFisher Superboost; 

AATbio STyramide;

Akoya Biosciences "Opal"

MANY MORE vendors (off patent a long time).

--

20240919H: ThermoFisher new Aluora Signal Amplification - "tyramide-like" radicals - also uses HRP polymers (i.e. HRPs-dextran backbone)-2ndAb) ... knowing ThermoFisher, some or all of the Aluora dyes might be Alexa Fluor dyes (see SuperBoost) under a new name.

20241015W: TermoFisher Spatial biology webinar stated Alexa Fluor SuperBoost is optimized for cells;

Aluora is optimized for tissues ... multiplex and sensitivity

I speculate that Aluora kits could use smaller HRP-polymers (i.ee. 10 HRPs inctead of say 100 HRPs per dextran polymer)

  10x-x100x

        $1 + $6 

potentially 

      $0.20 + $6 

if re-titer primary antibody (and test qualit of all other reagents)

TSA is more complicated (HRP polymer conjugated 2ndary antibody; H2O2 + fluorophore-tyramide) than 'standard' immunofluorescence methods, but might brighter - and when done optimally, essentally zero background.

IHC labs: those labs that routinely use HRP with DAB (and H2O2) COULD substitute the DAB solution with a fluorescent tyramide, for example, Alexa Fluor 488-tyramide (ThermoFisher / Molecular Probes). Best to be mindful of cost of reagents and that fluorescent tyramide is much more sensitive that DAB IHC. You will very likely need to re-titrate the concentrations (amount of volume of each reagent) onto the specimen of ALL your reagents, including the primary antibody (andHRP-polymer-2ndAb, etc). Any background from any reagent will increase background, which you may mis-interpret. At some point, you might even want to deliberately adjust the amount of primary antibody to NOT saturate binding to the antigen (ex. 50% of saturation), or more elaborately, make a series of specimens, dilution series of primary antibody, so understand the "binding curve". I am assuming/hoping your imaging pipeline is quantitative (a digital camera can be tested with a series of exposure times usinga bright, photostable specimen, such as 10 ms exposure ... series in 10 ms steps to 2000 ms [2 seconds]). tip: there are many places in a typical research fluorescence microscope where linearity can be lost (trivial example: photobleaching). 

 

ThermoFisher TSA pricing

20241029Tue prices - not logged in for JHU academic prices


https://www.thermofisher.com/order/catalog/product/A40001330
A40001330 Aluora 488 493/518 nm
$995.00 for 100 reactions (100 slides) (list price - not JHU academic price)
https://www.thermofisher.com/order/catalog/product/B40941
B40941 Sufficient for    50 Slides $407.65 
B40912 Sufficient for  150 Slides $791.65  = $5.28 each
(SuperBoost kit price 20241029Tue - online exclusive prices - may not be as good as academic price).
https://www.thermofisher.com/order/catalog/product/B40953?SID=srch-srp-B40953
B40953 Alexa Fluor 488 tyramide conjugate alone 495/519 nm $495.65 / 150 slides = $3.31 per slide (you supply all other components)
Aluora 488 TSA kit                                 $10.00 per slide
SuperBoost Alexa Fluor 488 TSA kit     $5.28 per slide

(assuming you use kit with standard protocol).
 

Additional info: ThermoFisher support emailed GM that Aluora is intended for multiplex and high sensitivity - check out primary Ab dilution series recommendation:


https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/protocols/multiplexed-tyramide-tissue-staining.html
We strongly recommend you optimize the primary antibody before using the kit. Positive and negative control slides should be stained with a serial dilution including 1:100, 1:500, 1:1000, 1:5,000, 1:10,000.

* GM: make sure you do "plenty of washes" after primary antibody (ex. 7 not "2 or 3") with large volume of buffer (cells or tissue sections in imaging dish, ~2 mL wash) and make sure specimen does not dry out -- that is keep submerged (not as big dilution: do more washes).

 

 

You can "quench" (aka kill) HRP enzyme activity with PeroxAbolish or other reagents / methods (heat for example), then do multiple rounds. See Berrry, ... Szalay, Taube 2021 Science, PubMed 34112666  (7plex Akoya TSA reagents and slide scanner). Tyramide signal amplification has been around since ~1990 (so long time off patent). 

* Tip from 1999: Brian Van Tine(at the time a MD/PhD student at UAB) at at CHLS in situ hybridization & immuocytochemistry course did 6plex tyramide signal amplification of PBMCs in solution (ex. CD3, CD4, CD8, CD20, CD64 plus one more),all very bright. I do not have the protocol details - probably spun cells down in a microfuge (cold rotor, balanced tubes) for wash steps. Very bright, very specific. I do not see references off hand but someone (1990s?) published tyramide flow cytometry. 

See (2023 cmmentary) https://pubmed.ncbi.nlm.nih.gov/37199234/ 

1997 research paper https://pubmed.ncbi.nlm.nih.gov/9071319/ 

TSA in flow (with some quantitation vs standard luorescent antibody labeling:

Clutter MR ... Nolan GP 2010 Tyramide Signal Amplification for Analysis of Kinase Activity by Intracellular Flow Cytometry. Cytometry A 77A: 10201031. Clutter references of prior paublications using TSA of cells in supsension:
23. Kaplan D, Smith D. Enzymatic amplification staining for flow cytometric analysis of cell surface molecules. Cytometry 2000;40:81–85.
24. Kaplan D, Meyerson H, Lewandowska K. High resolution immunophenotypic analysis of chronic lymphocytic leukemic cells by enzymatic amplification staining. Am J Clin Pathol 2001;116:429–436.
25. Kaplan D, Smith D, Meyerson H, Pecora N, Lewandowska K. CD5 expression by B lymphocytes and its regulation upon Epstein-Barr virus transformation. Proc Natl Acad Sci USA 2001;98:13850–13853. 26. Karkmann U, Radbruch A, Holzel V, Scheffold A. Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry. J Immunol Methods 1999;230:113–120.
27. Kaplan D, Meyerson H, Husel W, Lewandowska K, MacLennan G. D cyclins in lymphocytes. Cytometry A 2005;63A:1–9.

20240919H: ThermoFisher now has Aluora signal amplification "tyramide like" radical reaction,

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/immunofluorescence/aluora-spatial-amplification.html

8 dyes (some may be renamed Alexa Fluor dyes or Alexa Fluor PLUS dyes???)

1. 427/499 nm

2. 493/518 nm

3. 512/529 nm

4. 553/567 nm

5. 589/615 nm

6. 652/670 nm

7. 687/706 nm

8. 757/783 nm

Aluora Signal Amplification 202409 TSA like

Aluora & EVOS S1000 scope vs SuperBoost

Email from Allie Alcott, PhD, ThermoFisher, to GM 20241118M:

The Alouras have been optimized specifically for FFPE tissue, so if you're not using FFPE tissue or are staining cells, the Superboosts will be the better option. In terms of brightness, it's dependent on each channel. The Alouras were made to work for spatial imaging with our S1000, so they have matched intensity of each color. The Superboosts vary more (some are brighter, some are dimmer). When doing the unmixing for spatial imaging, having the matched intensity was imperative so the Alouras were created for that purpose. 

Let me know if there are any other questions or if that answered the ones you had!

Allie Alcott, PhD

Technical Sales Specialist – Imaging & Detection

EVOS / Plate Readers / Molecular Probes

Thermo Fisher Scientific

240-383-5432 | allison.alcott@thermofisher.com

EVOS Imaging Systems: www.thermofisher/EVOS

**

GM note; one way to look at Aluora is they are matched for the light sources and filter sets of the EVOS S1000.

Another way to to think of them as being either:

a) equally bright

and/or

b) equally dim.

Also the Aluora kits may have been tuned for tissue sections and/or may have simply increased price on the theory that customers will pay more for kits marketed for "Spatial Biology" (aka $patial Biology).

 

Conceptual         ?            ?$?

Ideally we would like an immunostaining and immuno-imaging method that produced quantitative data over the full dynamic range over the relevant biology. This is non-trivial. 

Imaging side: most modern scientific cameras for widefield and spinning disk cofnocal microscopy (sCMOS) are listed a 16-bit dynamic range, implying 0 .. 65535 "ADU" (analog to digital units") within one exposure. However, usually the camera offset is ~100, and at the high end, Poisson distribution noise is such that one would not want to come too close to saturation, so the dynamic range maybe ~100 .. ~59,000 (within ~10% of the pixel max), ~100 is ~7 bits (0..128), so realistically 16 - 7 - 1 = ~8-bit (albeit in wonderfully small steps). The best camera (and essentially most expensive) camera GM knows on the market in mid-2024 is the Hamamatsu ORCA-Quest2, with photon resolving range of 0 .. 200 (it also has a more sCMOS like ~100 - ~65535 readout mode). 

F-actin in the border of a 'typical' human cell (i.e. flat HeLa, U2OS, megakaryocyte) can be "lit up" with fluorescent Phalloidin (exclusively light up F-actin), and one could also express monomeric AausFP1 GFP-actin (G-actin monomers). Good luck seeing both G-actin monomers (in live cells they may diffuse the expected size of a 44 kDa monomer) and sub-plasma membrane F-actin bundles. 

Another examples are EGFR (erbB1) and Her-2 (ErbB2), which might be expressed at ~5000 molecule per cell (and mostly plasma membrane) of a normal human cell, but 500,000 to over 1,000,000 molecules for roughly the same size cancer cell (famously breast cancer, potentially glioblastoma and others). So adjacent normal and cancer cells might be 5000 and 500,000 molecule per cell, or for nearby plasma membrane pixels, say 1 and 1000 (if the plasma membrane was 1000 pixels or voxels). 

       

 

20240605W ThermoFisher Superclonal recombinant secondary antibodies ... available with Alexa Fluor PLUS fluorophores

* GM note(s): recombinant antibodies "should" perform better than "out of rabbit", "out of goat", out of donkey" etc, plus no more dead animals haunting you. Of course, ThermoFisher (and other rec Ab vendors) need to deliver conistently high quality manufacturing - and follow through with QA/QC. I do think they would do better by offering scFv-custom-optimized-tail, instead of just Fab and intact IgG.

from marketing info:

https://www.thermofisher.com/us/en/home/life-science/antibodies/secondary-antibodies/superclonal-secondary-antibodies.html

What are Superclonal recombinant secondary antibodies?

Thermo Fisher Scientific Invitrogen Superclonal secondary antibodies represent a recombinant antibody technology designed to provide precise and accurate detection of mouse, rabbit and goat primary antibodies in a variety of applications.

Our proprietary screening and production process yields specific mixtures of recombinant goat or rabbit secondary antibodies that bind with the epitope-precision of monoclonal antibodies, while also achieving the multi-epitope coverage (e.g., H+L) and sensitivity of polyclonal antibodies. Superclonal secondary antibodies are in vitro manufactured using synthetic genes after the first immunization. Each Superclonal secondary antibody is formulated and optimized to help achieve excellent results in ELISA, cell and tissue imaging (ICC/IF and IHC) and flow cytometry applications.

***

Superclonal plus ... 11 products  (as of 20240604U)

https://www.thermofisher.com/antibody/secondary/query/Superclonal%20plus

two examples (one IgG, one Fab):

Goat anti-Human IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor™ Plus 488

Goat anti-Human IgG Fab, Superclonal™ Recombinant Secondary Antibody, Alexa Fluor™ Plus 488

20240511A: 

Daniel Beacham, ThermoFisher - Molecular Probes (Eugene, OR) presented a seminar on 20250508W on fluorescent probes at the JHU SOM campus (Smith Bldg Atrium, Wilmer Eye Institute - attended by researchers from all over campus). Among the highlights:

* Dan is a coinventor of the Bacmam2 product line, Baculovirus derived 30 kb cargo transient transduction reagent, uses VSV-G to bind most mammalian cells (receptor is human, mouse etc LDL-R). After internalization, routed to endosomes/lysosomes, then escape to cytoplasm and cargo genes expressed from appopriate promoter(s) [the remaining bucolovirus vector genes are not expressed in mammalian cells - Bacmam2 is generated in insect cell lines]. The one major cell lineage Bacmam does not work in are hematopoietic cells, ex. hematpoietic stem cells (HSC), T-cells, B-cells, monocytes, neutrophils. Maybe TF-MPI will come up with a "Bacmam3" that does. Dan mentioned "2" binds ok, but do not escape from endosomes.

* Dan said Alexa Fluor Plus products vary in their 'tweaks' (my term) compared to 'regular' antibodies. Can be any (one?) of:

  • * tweak to fluorophore (possible methods include, not limited to, add sulfonylation to make more soluble; single isomer AF488).
  • * optimized linker (which is what I was informed when Plus came out - maybe marketing dept simplified story?).
  • * optimized conjugation site(s) -- that is choices of what amino acids on the antibody heavy and/or light chains (possibly by using non-canonical amino acids and 'click' chemistry; possibly adjusting reactive moieties, pH, etc, to get the fluorophores only onto specific amino acids ... possibly C-termini???).

* Dan alsoe said the key release criteria of Plus products is at least 1.5-fold better signal to background ratio compared to 'regular' secondary antibodies.

  • GM note: This implies to me that this makes "titering" each of the Plus antibodies -- and each primary antibody -- for each use may be crucial to see the full benefit of "Plus" prducts. As a further suggestion (by me), titering may need to be different for antigens on (or in) cell types with low expression vs medium or high expression. EGFR is expressed at low level on 'normal kidney' cells (i.e 5,000 EGFR per cell), much higher on some tumor cells (ex. 100,000 EGFR per cell) and very high on the classsic A431 lung cancer cell line (1,000,000 EGFR per cell, perhaps more on some cells -- for example a cell just before division would have ~2,000,000 molecules per cell, then divide to two cells each with 1,000,000 per cell [on average]). the 20-40 range of EGFR on these cells (we'll assume same surface area of each) might benefit from different doses of antibodies.

* I mentioned to Dan (post seminar) my (mis?)understanding of TF-MPI marketing "Plus" as 3x brighter than regular -- he disavowed that Marketing would ever put up such numbers, and remphasized the Signal-to-Background (SNB) 1.5x of more release criteria for each Plus product (he also mentioned not knowing prices). If 'only' 1.5x better, then if 1.3x fold more expensive, Plus would be a marginal win (if user takes the time to optimize titers of each reagent) -- but I suggest (hope) that in many cases "Plus" will be both brighter and higher SNB. 

 

20240307H:

Two fun facts (or at least colleagues repoorts):

To-Pro-3 DNA counterstain was so bright on Tim Feinstein's Leica SP8 that it destroyed a HyD detector. No additional details - so could have been "crazy concentration", "crazy laser power", "bad settings" (aka bad user), "user not following training", any or all of the above. The Leica HyD edetectors have a safety interlock - Tim told me zap was too fast.

Alexa Fluor 568 Phalloidin worked as a LIVE cells F-actin label for Dowlette-Mary. Normally users fix and permeabilize cells, so any Fluorophore-Phalloidin works. I encourage users to test AF568-Phalloidin on their (your) live cells and let me know if it lights the live cells up ... and that I should update this section with postive and/or negative results.

 

20240207W

--> see also (above) 20240605W "ThermoFisher Superclonal recombinant secondary antibodies ... available with Alexa Fluor PLUS fluorophores".

"Plan Plus" (see also Plan T below in this box for even brighter)

Alexa Fluor Plus secondary antibodies 3x brighter 1.3x more expensive

Famous quote: "Time is money".

ThermoFisher (Molecular Probes) introduced circa 2023 Alxa Fluor Plus secondary antibodies.

https://www.thermofisher.com/us/en/home/life-science/antibodies/secondary-antibodies/fluorescent-secondary-antibodies/alexa-fluor-plus-secondary-antibodies.html#table

Donkey anti-mouse is $355 for 1 mg (1 mL)

https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Highly-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A32766

If you use 1:100 dilution, then 10 ug (10 uL) is $3.55 per coverglass, imaging dish, etc.

the "standard" prouduct is 1.3 fold less, so $2.55.

the primary antibody would likely be used at the same amount, say $1 per specimen.

GM update #1 20240322F: ThermoFisher SuperBoost tyramide signal amplification is about $5.33 per coverglass, so with primary Ab at $1/coverglass would be $6.33 each coverglass (or 35 mm imaging dish), 10x to 100x higher intensity implying 1/10 to 1/100 imaging time.

Confocal microscope time is $27/hr (work hours, our Leica SP8 or Olympus FV3000RS).

User time making specimens and imaging: $0 from the standpoint of the P.I. = already accounted for.

So 1 hour confocal time:

SuperBoost:        $5.33 + $1 + 27 = $33.33.

AF### Plus:        $3.55 + $1 + 27 = $31.55.

AF### Regular:  $2.55 + $1 + $27 = $30.55.

Or, since Plus is 3x brighter, could reduce confocal time to 1/3 hour ($9) and Plus slide same brightness as regular for 1 hour:

Plus at 20 minutes: $3.55 + $1 + $9 = $12.55.

SuperBoost at 10 minutes (i.e. 1/6 imaging time): $5.33 + $1 + $4.50 = $10.83.

My recommendation is choose the same acquisition time, get "Plus" 3x brighter data, or SuperBoost 10x to 100x brighter.

SuperBoost is only 4 HRP per Antibody

https://www.thermofisher.com/order/catalog/product/B40943

Benefits of SuperBoost kits

Enhancement of signal using Alexa Fluor tyramides: SuperBoost kits utilize Alexa Fluor tyramides, which react with HRP to ultimately deposit bright and photostable Alexa Fluor dye on surrounding proteins and other similar molecules. SuperBoost kits are the only kits that combine the brightness of Alexa Fluor dyes with the enhancement of tyramide signal amplification to produce a superior signal.

Poly-HRP enhancement: Unlike TSA, SuperBoost kits employ poly-HRP-conjugated secondary antibodies. In such systems, several HRP enzymes are conjugated with short polymers, enhancing the signal by several fold over regular HRP systems. The poly-HRP is structured in such a way that the antibodies penetrate cells or tissue as efficiently as regular HRP-conjugated secondary antibodies. The molar enzyme/antibody protein ratio has an average value of '4'.

Reaction stop solution: Like any enzyme-based labeling system, it is possible to overdevelop the signal. SuperBoost kits include an HRP stop solution to halt the HRP reaction. HRP stop solution can be used to obtain maximum signal, without increase of background signal. Images produced with optimized HRP reaction times are as sharp as images produced with standard ICC/IHC/ISH methods, but with 10-200 times more sensitivity.

Reduction of background: SuperBoost kits include blockers for the elimination or reduction of endogenous peroxidase and fluorescent background signals. These blockers help ensure that only specific signals are enhanced while keeping non-specific/background signals in check.

//

GM: statement above that is is "possible to overdevelop the signal" - I suggest more likely the number of surface exposed tyrosines (phenylalanines, tryptophans, histidines are 'somewhat reactive' as well) will be limiting in TSA (or Styramide, see below) immunofluorescence experiments. 

SuperBoost 4:1 molar ratio:

HRP is 44 kDa

IgG is approximately 155 kDa (isoforms vary in MW: IgG1 vs IgG2a etc).

F(ab')2 is approximately 110 kDa  (without Fc)

So: 4 HRP (~176 kDa) + 1 IgG (~155 kDa) is ~331 kDa (I suggest ~333 as an easier number to remember).

I also note that Fitzgerald Industries sells HRP20, HRP40 and HRP80 conjugates (ex. to streptavidin, see also their product list for secondary antibodies) ... I believe number of HRP per polymer is 5x more than the number in the name, that is: HRP20-Streptavidin  has 100 HRP:SA, HRP40 has 200 HRP:SA, HRP80 has 400 HRP:SA. So, if access is not a problem (thin specimen or antigen/biotin on a coverglass or ELISA SBS plate surface) then HRP80 would be a whole lot of HRP molecules -- and bring in a whole lot of surface tyrosines to "dock" fluorescent tyramide or fluorescent Styramide (Styr below in AATbio section).

* as of April 2023, F.I. is now part of Biosynth,

https://www.biosynth.com/news/biosynth-agree-acquisition-of-fitzgerald-industries-international-from-trinity-biotech

example product pages (see website)

https://www.biosynth.com/p/65R-S103PHRP/streptavidin-poly-hrp20-conjugate

https://www.biosynth.com/p/65R-S112/streptavidin-poly-hrp40-conjugate 

Weird (if not DY-tyramide) and/or interesting: HRP40 with DY647 (DyLight 647)

     https://www.biosynth.com/p/65R-S128/streptavidin-poly-hrp40-conjugate-dy647

https://www.biosynth.com/p/65R-S105PHRP/streptavidin-poly-hrp80-conjugate

 

GM update #2 20240322F: AAT Bioquest now offers "Styramide" (Styryl-phenol aka styryl-tyramide - see freepatentsonline or google patents for patents), which they claim is superior to tyramide - see box below for their marketing info.

MAYBE Styramide will be superior to ThermoFisher Alexa Fluor ### Superboost TSA.

AATbio Styramide (styryl-phenol aka styryl-tyramide)

https://www.aatbio.com/catalog/power-styramide-signal-amplification-psa

Similar to tyramide signal amplification (TSA), PSA™ imaging uses the analyte-dependent reporter enzyme, horseradish peroxidase (HRP), to catalyze the covalent deposition and binding of labeled-Styramide™ substrates onto a target protein or nucleic acid sequence in situ. In the presence of hydrogen peroxide (H2O2), HRP converts labeled Styramide™ substrates into highly-reactive, short-lived Styramide™ radicals that rapidly bind to tyrosine residues on and proximal to the enzyme site. Styramide™ radicals have much higher reactivity than tyramide radicals, making imaging with PSA™ significantly faster, more robust, and sensitive than conventional TSA labeling. Since the added labeled-Styramide™ are deposited close to the HRP-target site, there is a minimal diffusion-related loss of resolution. PSA™ imaging technology can be readily added to any application that allows for integrating HRP into its protocol. Such applications include IHC, ICC, IF, in situ hybridization, and ELISA.

GM update #3: (minimal info here); a Japanese group has published several papers using "methyl-Luminol" as an alternative to tyramide for HRP. See pubmed or ask GM for more info.

**

I suepect many experiments could use lower concentration of secondary antibody to get "same or almost as bright" as the common 1:100 dilution = same more money, similar brightness. There is also potential to optimize incubation time of the primary and/or secondary antibodies, such as dilute further, incubate 24 hours (aking sure the specimens do not dry out), see (direct label flow cytometry experiments:

Whyte, C. E., Tumes, D. J., Liston, A., & Burton, O. T. (2022). Do more with less: Improving high parameter cytometry through overnight staining. Current Protocols, 2, e589. doi: 10.1002/cpz1.589

***

"Plan T" for 10x to 100x brighter:

Big boost in signal: tyramide signal amplification (TSA) has been available since around 1990. Used optimally, can increase signal approximately 100x, while background is still close to zero. This could reduce imaging time by a lot (33 fold compared to "Plus" above) or increase signal for same scan time, or some combination. HRP is "easy to kill" the enzyme (Biocare Medical "PeroxAbolish" is one of the coolest name products for this), then multiplex,

Many companies now offer TSA reagents, see for examples (two of many):

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/immunofluorescence/tyramide-signal-amplification-tsa.html

https://www.tocris.com/product-type/tyramide-signaling-amplification-tsa 

 

McTip 20240131 Centrifuge down antibodies

McTip 20240131 Centrifuge down antibodies (minifuge, 4 C rotor)

GM note1: use cold (stored 4 C) microfuge rotor - if use room temperature rotor, you will cook your antibodies - similar to frying egg whites. You could use ultrafiltration (i.e. 40nm Amicron filte) though some risk of your antibodies sticking to the filter (lose of titer).

GM note 2: Also, if you use BD/BioLegends Brilliant Violets (BV421, etc), Brilliant Ultraviolets (BUV395 etc) you should use BD's Brilliant buffer to avoid aggregation of the Brilliants; quantum dots may or not play well in Brilliant Buffer and vice versa. I also note the authors used several QDot antibodies - quantum dot antibodies originally (first generation thermoFisher/Molecular Probes, acquired QDot Corp circa 2005) were highly prone to aggregation - may or not have been corrected in current (2024) versions. QDots have "blinking issues" which may be good for PALm/STORM/SMLM super-resolution microscopy and/or bad for standard "brighter is better" fluorescence microscopy.

Fun fact: Authors used many Brilliants (BUV, BV, BB) and several quantum dots. Brilliants technology won Nobel Prize in Chemistry in 2000 "conductive polymers" (see Sirigen history page - acquired by BD); Quantum dots won Nobel Prize in Chemistry in 2023 (more for light sources for televisions and computer screens than for fluorescence). 

20240806Tue update: now published in Cytometry A - centrifuge details (though no mention of whether the minifuge rotor should be kept refrigerated):

https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.24841

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells

Andrew J. Konecny, Peter L. Mage, Aaron J. Tyznik, Martin Prlic, Florian Mair

Cytometry A Volume1

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