#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.

**

more on #1.5H coverglass thickness - nice blog

THE IMPORTANCE OF THE CORRECT COVERGLASS THICKNESS FOR PHOTOMICROGRAPHY
Robert Berden (Motic Microscopes) - Posted by Motic America on January 17, 2023
https://moticmicroscopes.com/blogs/articles/the-importance-of-the-correct-coverglass-thickness-for-photomicrography

Partial text:   

Measuring Cover Glass Thickness  --- It is simple to measure coverslip thickness with a digital micrometer.  I use a Neoteck digital micrometer with a resolution of 0.001 mm (cost $50 online).  It is impractical to measure coverglass thickness routinely unless one is performing high resolution measurements or trying to detect single molecules. 

GM note - two alternatives to calipers (calipers have the advantage of simplicity and speed -- I would not trust the manufacturers claim of "resolution of 0.001 mm = 1 um)

A) can "stand up' one or more coverglasses and image with 10x or 20x objective lens. I note the edges are not smooth.

B) image through a coverglass, recognizing the Z-drive values (ex. 0.000 mm --> 1.234 mm) are movement in air, whereas the coverglass is refractive index 1.518. Can put on both sides sharpie marks (black or other color ink "magic marker") or diamond scribe scratches, to make easy to see surfaces. 

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).

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.

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.

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).

***********

  * 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). 

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.

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.

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 (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 ultrafil

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PTI QuantMaster name is missing an "a", really PTI QuantaMaster spectrofluorimeter. We are sticking with QuantMaster 1 and...

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