Re: immunostaining of melanoma samples/melanin bleach

The way I always did melanin bleach (at least after 1975 since from
1967-1974 I also tore my hair out with most others) is the following.  This
is not my invention!!  Learned it at AFIP in 1975 and the tech their said
they learned it from someone else so have no idea who in antiquity gets
     Do the bleaching of the melanin while still in the paraffin ribbon.
So when you ribbon, do not put the ribbon on your heated water bath instead
put ribbon right on a small container (like a Tissue Tek upright staining
container) that contains permanganate.  Length of time empirically
determined.  I think we did maybe 10-12 minutes as I remember back.  Then
pick up section with uncharged slide and refloat on another small container
of oxalic and section still in paraffin will turn from ugly brown to
opaque.  Then with uncharged slide refloat on your water bath and let
section relax and flatten which is important.  Then pick up on charged
slide.  If you want to be fastidious about your experimental procedure, cut
ribbon for "no bleach" and float on cold distilled water same amount of
time you did for the "bleach section" and then transfer to water bath and
pick it up.  Now you have two, nice, flat sections; one bleached and one no
bleach.  Dry and stain as normal.  Once I started doing this, I never again
feared a melanin with and without bleach request.

Immunex Corp.
Seattle, WA

                    Amos &                                                                    
                    Theresa              To:                          
                    <atbrooks@sne        cc:     HistoNet Server                              
          >               <>                       
                                         Subject:     immunostaining of melanoma samples      
                    12:50 PM                                                                  

    I think the problem you are experiencing is more likely melanin
Just to be sure, don't stain a section at all. Deparaffinize and hydrate,
counterstain it, dehydrate clear & coverslip it. I think you will probably
still see this background. You could try a different color chromagen, or
bleaching the sections if this is the problem. Bleaching the sections can
done with potassium permanganate & oxalic acid, or a concentrated peroxide
solution. Be warned however that sections love to fall off during these
    By the way if anyone has success with bleaching WITHOUT knocking tissue
the slides, please post your incantations, spells prayers or other tricks
make it possible! It's hit & miss for me at this point.
Amos Brooks

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