Re: Fluorescent immunohistochemistry

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From:"tony.whyte" <> (Reply requested), (Reply requested)
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Hi Jennifer,

I have answered each of your questions in turn (where I have been able!).

>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?

Should do.

>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?  

Usually less sensitive, so you would need to increase the concentration of

>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?

Yes, the ABC method is an immune complex method and very 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

you won't need levamisole or H202, as you are not using these enzymes, but you
will need the serum blocks.

>4.  How long do you "develop" or incubate the fluorescent tag?  Does leaving
>it on longer give background like leaving DAB on too long?  
1 hour at RT in the dark is generally OK, or overnight at 4oC.

>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?  

No, but you would probably want to use an anti-fade mountant.

>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?

Depends on your filter sets. FITC and TRITC (or Texas Red) are most commonly
>7.  What are some of the major problems that I might encounter with
>fluorescent labeling? 
Too many to list!

>8.  Are there methods to quench autofluorescence in tissues? 

There are several, but do not fix in gluteraldehyde! (Paraformaldehyde or
acetone/mthanol are OK).

Hope this helps.  If want further info, contact me directly

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