Re: [Histonet] Antibodies for FACS and IHC

From:Sharon Cooperman

You can use antibodies that are routinely used for FACS for IHC and 
visa versa.  Most FACS usable antibodies I have tried that work with 
formalin fixed cells on FACS (eg. you fix the cells first) will work 
at least on frozen sections, sometimes on FFPE sections.  Sometimes 
antibodies that work on one method have too much background or not 
enough signal on the other method.  You don't need to have a 
fluorescence tagged primary for FACS and sometimes it helps to 
increase your signal by using a secondary antibody.  Sometimes a 
fluorescent primary is enough signal for immunofluorescence and 
sometimes not, but you can always use a fluorescent secondary (tagged 
with the same fluor) with a tagged primary.  But, I don't have any 
hard and fast rules (other more experienced people may) I just try it 
and work it out for each antibody as the need arises.


>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
>Histonet mailing list

Sharon Cooperman       	     
NIH, NICHD, CBMB                     301.435-8417
Building 18T, room 101               301.402-0078 fax
Bethesda, MD 20892

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