Fluorescent immunohistochemistry

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From:"Johnson, Jennifer(Hist)" <Jennifer.Johnson@genzyme.com>

Hi Histonetters!
	 For the past several years when doing immunos, I have used alk phos
and peroxidase systems using ABC kits from Vector.  Now I have a need to
double label the same cell(s) (in both frozen and paraffin)in the same
tissue section. I have tried every color chromagen combination in the book
(DAB, DAB + Ni, AEC, Vector red, Vector Black, Vector Blue, Nova red...)
with lots of help from Vector :-).  Because I want to demonstrate the
presence of an experimental drug and then use another antibody to
demonstrate the cell type of that cell, I have given up on precipitating
chromagens and have set my hopes on fluorescence.  I am having difficulty
finding information and protocols for immunofluorescence. I have surfed the
web and found very minimal information. I have searched our histology texts
(luckily we have several)and only one, Sheehan's Theory and Practice,
discusses it in any length.  I was hoping that you all could help me out by
answering a few general questions that the texts have not helped me answer.
And, maybe find it in your hearts to send me a few tips, pearls of wisdom or
protocols to help me figure out where to begin!!

1.  I have worked out the concentration of antibody needed for both paraffin
and frozen tissues with the ABC systems.  If the antibody works for alk phos
and peroxidase, will it also work for fluorescence?

2.  Will the dilutions for the antibody be about the same or is there some
difference in the inherent in either the precipitating chromagen system and
the fluorescent system that makes one system work at a lower dilution?  

2.  I am thinking about using a secondary that is already fluorescently
tagged, but without the amplification of the biotin system, is this type of
fluorescence system less sensitive?

3.  Are the needs for blocking different?  
	Using the systems from Vector, depending on the antibody, I had to
perform some/all blocking listed below which differed per kit:  
		Alk Phos - Levamisole, Normal goat/horse serum and
		Peroxidase - peroxidase quench, Normal goat/horse serum and
		Sometimes in either system I had to use an Fc receptor block

4.  How long do you "develop" or incubate the fluorescent tag?  Does leaving
it on longer give background like leaving DAB on too long?  

5.  Are there any restrictions on which buffers or solutions that may quench
the fluorescence or otherwise ruin the experiment?  Are there any
recommended buffers or solutions that I have to use to obtain staining?  

6.  Any suggestions on which tags work best?  I have been reading about
Alexa dyes and was thinking of using two that were far enough apart, Alexa
488 and 594.  Any experience with Alexa dyes?
7.  What are some of the major problems that I might encounter with
fluorescent labeling? 

8.  Are there methods to quench autofluorescence in tissues? 

I realize that this is a lot to ask.  If there is a book or article that you
could recommend, I would love to hear about it!  I would be eternally
grateful for any information that you all could provide.  This, by the way
would make a great topic for an NSH meeting seminar.... 

Jennifer Johnson
Principal Research Associate
Genzyme Corp.

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