methyl green/frozen IHC

From:meadows <>

Date: 20 Sep 2000 11:12:39 -0500
   Subject: Methyl Green

   Does anyone have the recipe for methyl green, to be used as a counterstain 
for tunel staining?
   Thanks in advance

Here's a recipe which works well for me:

            0.5% (w/v) Methyl Green disolved in 0.1M sodium acetate
              - pH to 4.0

1. After application of final substrate and washing of slides, immerse in 
Methyl Green solution for 10 min.

2.  Wash slides in 3 changes of dH2O:  dip rack 10 times in first and second 
washes followed by a 30 second immersion in the third

3.  Immerse slides in 3 changes of 100% N-butanol:  dip rack 10 times in first 
and second changes, followed by a 30 seconds in the third

4.  Clear sections in 3 changes of Xylene - 2 minutes each

Date: 26 Sep 2000 22:46:12 -0500
        From: "Jim Manavis" <>
        Subject: Frozen IHC

        Can someone tell me their experience with frozen sections which are 
        fixed in cold acetone prior to applying immuno histochemistry.
        Which procedure do you find is superior? Does it also vary depending 
on         the Ab used?
        Post fix immediately, dry, leave them say, overnight in the -20 
freezer,         and then apply IHC? or
        Do you air dry after sectioning, store overnight, then post fix just
         prior to IHC? or
        Do you section, post fix and then apply IHC on the same day?

        Thanks, Kathy Cash

With my frozen sections I allow them to reach room temperature on the benchtop 
(about 30 minutes).  They are then immersed in cold acetone (-20 C) for 5 
minutes.  They are transfered immediately to 1X PBS and washed 3 times.  At 
this point I will quench for endogenous peroxidase - if necessary.  If not, I 
go right ahead with the IHC.  I do find that the post fixation is dependent on 
the particular antibody used.  so, I always try several different post-fix 
methods when tweaking a new antibody.

Hope this helps,

Angeline Martin
Cell Biology and Anatomy
Medical College of Georgia

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