frozens, superfrost plus charge, fixation

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From:Gayle Callis <>
Date:Mon, 05 Apr 1999 11:12:31 -0600

Often the problem with mouse tissues, on super frost plus charge slides,
is the peroxidase blocking step.  The bubbling will lift the sections.

You can air dry several hours, fix with cold acetone 10 min, air dry for 15
min, fix again in acetone for 10 min, air dry for 1 hour or longer.
Minimum air dry before fixation is 30 minutes.  Some people fix for 10 min
in cold 4C acetone, it may work well for you, I prefer the double fix, for
better morphology and section retention.  If you are suffering from lots
of humidity, put your slides in a dessicator containing silica gel, to have
a drier atmosphere.  

Use Dako peroxidase blocker (0.03% hydrogen peroxide) very gentle, and 
designed for frozen sections (PharMingen recommendation).  Anything
stronger that this hydrogen peroxide has chewed my sections off the 
slide.  Avoid methanol, this will interfere with the immunostaining
of B and T cell markers, or you can make up your own blocker with 0.03%
hydrogen peroxide for 5 - 10 min time, in PBS.  I prefer the tidy Dako, as it
also contains some sodium azide, very convenient!

Gold plus slides are optimized for acetone fixation only, the Super frost
Plus Charge ( a trademark of Erie) works well.  If you buy the Fisher
brand of Plus Charge, they are cheaper than BD (Found out Erie makes their
slides!).  That Plus charge is the important thing, as this is an Erie
exclusive trademark.  

Hope this helps
Gayle Callis  

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