Re: Fluorescent immunohistochemistry
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From: | "tony.whyte" <tony.whyte@bbsrc.ac.uk> |
To: | Jennifer.Johnson@genzyme.com (Reply requested), histonet@pathology.swmed.edu (Reply requested) |
Reply-To: | |
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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
antibody.
>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
>avidin-biotin
> Peroxidase - peroxidase quench, Normal goat/horse serum and
>avidin-biotin
> 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
used.
>
>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
(tony.whyte@bbsrc.ac.uk)
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