[Histonet] Long reply to Re: Antibodies for FACS and IHC

From:Gayle Callis

First of all what species tissues/cells are you going to be working with, 
mouse?  Rat?  Human?  We use the same antibodies for both FACS and IHC.
First of all, when you are purchasing anitibodies, as from BD Pharmingen, 
they tell you what applications are recommended in their catalogs - little 
abbreviations. Be sure to access the specification sheets on their 
websites, these are filled with info, and even references to publications 
for any given antibody.  You have to do a bit of sleuthing - a bit of work 
but fun too.

Some of our combinations are

1.      purified antibody ( we also use monoclonal antibody supernates from 
cell lines producing antibodies, grown up in house)
         secondary antibody conjugated to fluorophore

2. although infrequent FACS, but a possibility
         Purified antibody
         Secondary antibody-biotin
         Strepavidn-Alexa dye conjugate

1.      Purified antibody
         Secondary antibody -Biotin
         Strepavdin-HRP or AP

2.      Purified antibody
         Secondary antibody-HRP or AP

Our favorite is and works very well  murine IHC when the primary is a Rat 
antiMouse monoclonal .

1.  For FACS and fluorescence microscopy
         Biotinylated primary
         Strepavidin -Alexa fluorophore conjugate (499 = FITC, 555 = 
rhodamine) for FACS or fluorescence microscopy
2.  For IHC, use Strepavidin-HRP for peroxidase IHC or Strepavidin-AP if 

a. For FACS
         Primary antibody-conjugated to FITC - can use this alone for FACS

b.      For IHC
         Primary conj FITC
         Rabbit antiFITC (DAKO)
         Donkey antiRabbit-HRP
If the primary is conjugated to PE, you can even buy antiPE and do IHC.

Most companies have recommended dilutions of their antibody (ug/ml) for 
FACS, however for IHC you would have to do a dilution panel to determine 
your working concentration for any given fixative, paraffin processing, 
retrieval etc.  Most companies will test antibodies for frozen section use, 
but not always for paraffin embedded tissues.  Dilution panel target 
concentration can start at 10 ug/ml, and then you dilute out.  Basically 
you will do the testing based on what they already tell you, be prepared to 
do some work.
Best sources of fluorescent secondaries - some companies that are 
excellent, and there are others.  It is advisable that the secondary 
antibody be adsorbed against the species being stained.  If you use a 
biotinylated primary the negative control must be host of primary IgG or 
isotype matched IgG that is also biotinylated.   You can purchase all 
different species IgG conjugated to biotin, FITC, etc, etc from Jackson and 
other sources.

         Southern Biotechnology - excellent antibodies for murine work.
         Jackson ImmunoResearch
         BD PharminGen

If you want to try secondary antibodies conjugated to Alexa fluorophores, 
Molecular Probes, they are available, we do not use these  OR you can buy 
an Alexa fluor conjugation kit and make your own secondaries, particularly 
if you have a company preference for buying secondary antibodies.

One thing to keep in mind, there are now primary antibodies conjugated to 
Alexa fluorophores, and you probably would have to buy antiAlexaFluor 
secondary from Molecular Probes, I have not done this.

I have never had problems using primary antibodies for FACS and then IHC 
application. You will have optimize your staining kit/system for the 
antibody you are using.  Just do the dilution panel on the primary, as kits 
are often very standardized to help you out there.

We have a protocol for biotinylated Rat antiMouse CD4, (0.5mg/) diluted out 
1:15,000 and fading at 1:20,000 for 30 min
Strepavidin-HRP 1:500 dilution of 0.5mg/ml, for 20 min
DAKO DAB+, followed by their DAB enhancer.
There was some special blocking done prior to staining, and not the same as 
some else might do it.
If someone else was using this antibody as we do, their Strepavidin-HRP may 
not be as effective as mine, nor will someone elses DAB chromogen.   And 
they may not have the same working dilution as we used for the primary.

You can go to company websites which provide staining protocols on how THEY 
do their frozen section work, etc and tweak the method to suit your needs.

There are basic rules but no hard and fast rules on how you do IHC with 
antibodies used for FACS, Histonet abounds with answers, so if you have a 
particular antibody in  mind, use Histonet Archives for a search 
www.histosearch.org, and if you don't find it, ask.  Use publications also, 
they should have information in methods and materials, invaluable to see 
how others have done it, plus it usually provides sources of 
antibodies/secondaries, etc all reagents used for both FACS and IHC.

I suggest you get the Molecular Probes 10th Edition handbook, they will 
send it to you or you can access it online from their website.  This book 
is very educational for fluorophore usage and also just understanding some 
of the chemistry of these fluorescent dyes.

Good luck, sorry this was so long

  03:26 AM 5/26/2005, you wrote:
>Please can anyone help?  How are antibodies produced for different
>applications different?  Is it possible to use antibodies that are
>currently being used for IHC, for FACS analysis?   We currently have
>unconjugated primary antibodies which we use for IHC, could we use a
>fluorescent secondary antibody to then use it for FACS.   If I wanted to
>source a new antibody that could be used for IHC (probably frozen sections)
>and FACS - do I need to be sure it's been tested in both applications or
>can I make a judgement based on it's performance in just one of the
>applications?  Is it best to source a fluorescent-conjugated primary?  Or
>will this mean there won't be enough amplification for the IHC application?
>  Sorry for all the questions but I'm venturing into new territory!!
>Thank you in advance,
>Nicola Cragg BSc
>Epistem Ltd
>Manchester, UK

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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