[Histonet] Quick frozen sectioning and IMHC questions

[Lewis, Patrick]

Hi everyone,

 

I cut my first frozen sections, human tonsil, and I have stored them at
-70.  When I take them out to use, should I bring them to -20C and fix
them right away?  Or should I bring them to room temp and then pap
pen/wash/block/wash them then fix them before adding my primary
anti-body.

My original plan was/is to bring them to room temp, pap pen them and
then wash/block/wash fix.  I am asking for your advice as my boss is
away for two weeks and the protocol he left me with is a bit vague and
requires some modifications to fit the research that we are doing.

I was just wondering if there is a compelling reason to keep the
sections at/near -20 after sectioning up through the fixation before
starting the Immunohistochemistry.

Also, we are using BSA as one of the components in our blocking solution
as well as a component in our wash solution.  Is there a danger in using
lower/higher quality BSA?   I'm a little worried that our BSA might
contribute to increased background if its quality is not high enough.

The BSA we may use is A-8327 Sigma, and I am thinking this might be
better A3294, or one of these (A9647,A7906,A6793)

This is a new job for me, and its been a long while since I have done
any Immunohistochemistries.so I am going over the reagents we have on
hand while I refresh my memory on immunohistochemistry and sectioning
techniques.

 

Thanks for all your support

 

Patrick

 

 

Patrick Lewis

Research Associate II-Bench| Infections and Prematurity

Seattle Children's Research Institute

206-884-1115  OFFICE 

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000-000-0000  CELL 

206-884-7311  FAX

patrick.lewis <@t> seattlechildrens.org

OFFICE  1900 9th Avenue Seattle, WA 98101

MAIL      M/S C9S-8, Seattle, WA 98101

WWW     seattlechildrens.org <http://seattlechildrens.org/> 

 



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