[Histonet] querry [SDS and IHC]

[John Kiernan]

That's a useful reference (Brown et al., 1996). Thanks 
for sharing it. I'm probably being a bit fussy, but 
treating frozen sections or cell cultures with a
detergent isn't really antigen retrieval, despite the
title of the paper. The SDS makes holes in the cell
membranes, so that the big antibody molecules can 
get inside. Triton-X (a non-ionic detergent) is
often used for this purpose on thick free-floating
frozen sections of minimally formaldehyde-fixed brain.
-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
May Wei wrote:
> 
> Please visit
> http://www.ihcworld.com/_protocols/epitope_retrieval/frozen_section_ar.h
> tm
> 
> Antigen Retrieval Method for Cryostat Frozen Tissue Sections
> 
> Description: This is a simple method for antigen retrieval on
> aldehyde-fixed cryostat tissue sections or cultured cells. In many case,
> a brief 5 minutes pretreatment with 1% sodium dodecyl sulfate (SDS)
> produced a dramatic increase in staining intensity by
> immunohistochemistry and immunofluorescence.
> 
> Solutions and Reagents:
> 
> 1% Sodium Dodecyl Sulfate (SDS) in PBS:
>       SDS --------------------------------------- 1 g
>       0.01M PBS (pH 7.4) ---------------- 100 ml
>       Mix to dissolve.
> 
> Procedure:
> 
> Rinse sections three times for 5 min each in PBS.
> Cover sections with 1% SDS solution and incubate for 5 minutes at room
> temperature.
> Rinse sections three times for 5 min each in PBS. It is important to
> wash sections well, otherwise residual SDS will denature the antibodies
> subsequently applied to sections.
> Incubate sections in serum blocking solution.
> Incubate in the primary antibody and complete immunohistochemical
> staining steps as desired.
> 
> References:
> 
> Brown D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, Alper S
> (1996)  Antigen retrieval in cryostat tissue sections and cultured cells
> by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol.
> 105(4):261-7. PubMed Abstract
> 
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Arvind
> Sent: Tuesday, September 27, 2005 1:22 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Cc: arvind <@t> nbrc.res.in
> Subject: [Histonet] querry
> 
> can any one please tell me how to do antigen retrieval on soft tissue,
> what i do is i took cryosections on slide for human fetal brain  tissue
> age upto
> 7 months (prenatal) sections are 40 micron thick, when i do HIER these
> sections are comming off the slides at some points, i use gelatin
> subbed slides for this  please guide what to do in detail
> 
> thanking in advance
> 
> Arvind Singh Pundir
> 
> National Brain Research Centre
> gurgaon, Haryana, INDIA
> arvind <@t> nbrc.res.in
> 
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