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

  * 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.

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 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 Volume105, Issue6 June 2024 Pages 430-436 (online April 2024). 

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.14.571745

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells
Andrew J. Konecny, Peter Mage, Aaron J. Tyznik, Martin Prlic, Florian Mair

Antibody mixes were centrifuged for 10,000 x g for 5 minutes immediately prior to use to remove antibody aggregates. We have observed a decrease in aggregation of Qdot 605 :: CD2, BUV615 :: CD141, Qdot 625 :: NKp46 (CD335), Pacific Blue :: CD1c, NovaFluor Blue 555 :: CD8a, NovaFluor Blue 585 CD4, and RB744 :: CD127. An example for such aggregates before and after centrifugation is shown in Online Figure 18.

Apple Mac users networking SMB://networkname is equivalent to Windows PC \\networkname

(the above line item is from McTips 2018 index).

for example, if a server is \\TikiGoddess on a Windows PC, then SMB://TikiGoddess on a Mac.We do not currently have a PC or server called TikiGoddess - to see Tiki_Goddess, see http://confocal.jhu.edu/gallery   .

Note: You may need to be logged in as a local user, not any JHU SOM Domaion login (ex: WIN domain). Local login cannot see the Domain assets for security. You can easily log in/out or "switch users" between a local and a Domain login.

Please do not give out the names of our file server or any image core workstations to anyone.

**

You can create a network "share" on YOUR PC. Your office/lab PC is owned by JHU, and managed by your PI, so you should get permission before creating a "shared" folder -- and if you do not fully understand networking and computer security you should probably NOT crete any shartes.

For example, C:\TikiGoddess\Share could be made visible on the campus network (may be restricted to being on -- or not on -- the Domain). Then you could transfer files from our acquisition PC to your PC "share", and as soon as you get back to your lab, move the contents from your "Share" folder to a secure (not visible not network) folder. 

We recommend you upload from our acquisition PC(s) to your JHU OneDrive - this is simpler (though usually slower) than network share drives. MyJHU --> Cloud --> JH OneDrive (can be made a favorite in MyJHU), upload your new data (i.e. save to a folder with today's date, so your new data is segregated from previous data), then sign out of your OneDrive, wait for browser page to update, then sign out of MyJHU, wait for browser page to update, then close the web broswer.

Reminders:

* No USB drives on any of our image core PCs.

* no checking your email (JHU or personal) on any of our PCs. Besides your privacy, if you get an email with a computer virus it could infect our PCs and JHU network (which could lead to you being fired for cause). 

* No surfing the "Dark Net" on any of our PCs - and please use MyJHU just to access OneDrive (not email, not other content ... not your daily covid-19 "click"). 

McTips 2018 PDF (as of 201810002) go to: McTips 2018 

https://works.bepress.com/gmcnamara/84

The McTips 2018 includes some of my thoughts with respect to Fast Photon Counting (FPC) to make fluorescence confocal microscopy both faster and more quantitative than is now practiced by most biomedical researchers (i.e. twiddle the HV gain and offset values until someone proves their boss' hypothesis ... especially when they are using 'Santa Crap' antibodies and don't bother with controls).