[Histonet] Re: methyl green counterstain

[Osborn, Barbara]

I believe it's the antigen retrieval that affects the methyl green
counterstain.  We purchase our methyl green from Vector Labs and
although the staining is lighter than sections with no antigen
retrieval, it does not  fade out during dehydration.  We counterstain
for 10 min at 60oC with decent results.

 

Barbara Osborn

University of South Florida

 

Message: 6

Date: Thu, 25 May 2006 17:50:03 -0400

From: "Jacqui Detmar" <detmar <@t> mshri.on.ca>

Subject: [Histonet] methyl green counterstain

To: <histonet <@t> lists.utsouthwestern.edu>

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Hi all.  I am doing some immunohistochemistry and TUNEL staining on
mouse placental tissue.  I have noticed that my methyl green
counterstain on placentae assayed for TUNEL looks normal, but when I
apply the same steps to tissue from the same block, but exposed to
immunohistochemistry, the methyl green counterstain is very weak,
bleaches out quickly during dehydration and generally looks pretty
crappy.  The methyl green recipe I am using is as follows:  0.5% methyl
green in 0.1M sodium acetate buffer, pH 4.2.    I would like to
emphasize that no matter how quickly I dehydrate with the IHC sections,
the colour is still very dim.  

 

The reason I am using methyl green is b/c I am doing IHC for nuclear
proteins (Ki67, etc.) and don't want to use hematoxylin, since it's a
little dark.  Any suggestions?

 

Thanks,

 

Jacqui Detmar