Re: Re[2]: Immunohistochemistry

From:Gayle Callis

In general, we tend to not do a lot of protein blocking with IFA, but do
make sure the secondary antibody is adsorbed to the species being stained,
do a dilution panel on secondary fluorochrome conjugate, and also use
F(ab')2 fragments of IgG secondaries conjugated to fluorochromes.  

For FACS and IFA on sections, the Alexa fluorochromes are unparalleled for
results, I agree with Ronnie. You can buy them as strepavidin conjugates
(be sure to do avidin/biotin block), or conjugated to antibody, even kits
to do your own conjugates from Molecular Probes. 

By background, garbage, staining on tissues.  Remember the signal of your
fluorochrome should be much brighter than nonspecific background, hence
brighter fluorochromes help.  If the cells stain well with fluorochrome, we
tend to ignore much of the background (dull greenish color) since cells
stained with Alexa 546 or 488 are brighter. There are some background
stained things that just don't go away.  

At 10:08 AM 4/12/02 -0400, you wrote:
>     I'd second what Tim says, and add that if background is still a problem
>     after using casein (non-fat dried milk) you could add either gelatin
or PVA
>     (both 2%) to your blocking solution.
>     I would also like to mention to anyohne doing IF that, in Dallas, we
>     switched all our fluorescent secondaries to Alexa dyes (Molecular
>     as we get a much brighter signal,less fading on exposure to UV, and less
>     background. In fact we exposed a section with Alexa 594 (the
equivalent to
>     Texas Red) continuously for 48 hours with very little fading of the
>     reaction (unpublished results).
>     Ronnie Houston
>     Regional Histology Operations Manager
>     Bon Secours Health Partners Laboratories
>     5801 Bremo Road
>     Richmond,VA 23226
>______________________________ Reply Separator
>Subject: RE: Immunohistochemistry
>Author:  "Morken, Tim"  at BSHSIBTW
>Date:    4/12/02 9:15 AM
>       Solutions of  powdered milk have been used (the protein casien
>       (sp?)blocks
>       non-specific binding)and some companies sell their own
>       proprietary
>       protein-free blocking agents.
>       Tim Morken
>       CDC, Atlanta
>       -----Original Message-----
>       From: Miller, Barbara []
>       Sent: Friday, April 12, 2002 8:48 AM
>       To: ''
>       Cc: Miller, Barbara
>       Subject: Immunohistochemistry
>       Does anyknow of blocking agents other than serum or albumin
>       that would
>       provide a low background for immunohistochemistry.   We are
>       using a
>       fluorescent  dye (often Texas Red) and background is a problem so far.
>       Thank you for your help.
>       Bobbi Miller
>       Department of Gastroenterology
>       Roudebush Medical Center
>       Indianapolis, IN
>       e-mail:
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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