[Histonet] Long reply to Re: Antibodies for FACS and IHC
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
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
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)
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
Southern Biotechnology - excellent antibodies for murine work.
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
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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