RE: Avidin/Biotin blocking
Dear Fernando,
After the antigen retrieval step!
In non-antigen retrieval treated FFPE tissue sections there is no
detectable endogenous biotin, probably due to fixation/embedding. Realize
that antigen retrieval methods may reveal endogenous biotin again,
especially when using Tris/EDTA pH9.0.
Apart from the streptavidin-biotin pitfalls mentioned by Gayle Callis, I
would like to add another pitfall that should be regarded as "endogenous
(strept)avidin-like". Van den Oord et al. described the specific binding
of biotinylated secondary reagents to hepatitis B surface antigen infected
cells in Bouin- or formalin-fixed human liver (J Histochem Cytochem 37:
551-554, 1989). This "endogenous (strept)avidin" is probably not blocked
effectively by regular endogenous biotin blocking methods.
Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands
Fernando Capela e Silva wrote:
>Date: 14 Apr 2003 12:36:47 -0600
>From: Fernando Capela e Silva
>Subject: Avidin/Biotin blocking
>
>In general immunohistochemistry protocol where should I use avidin/biotin
>blocking?
>
>1. Peroxidase quenching solution
>2. Pre-treatment/Antigen Retrieval
>3. Serum blocking solution (10% non-imune serum)
>4. Primary antibody
>5. Secondary Antibody
>6. Streptavidin/Peroxidase
>7. Substrate/Chromogen
>8. Counterstaining
>9. Mounting
>
>Best Regards
>
>Fernando Capela e Silva
>Laborat#243#rio de Biologia da Conserva#231##227#o
>Departamento de Biologia
>Universidade de ...vora
>Apartado 94
>7002-554 ...vora
>PORTUGAL
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