Re: Counterstaining after in situ hybridization


What we do routinely in NTB2 emulsion-dipped brain sections after ISH with
35S is to use thionin as a counterstain after the slides are developed in
D19.  An excellent protocol for this can be found at:
Thionin is also a Nissl stain though, is it not?  True, in a protocol for
ISH with an oligonucleotide probe, rather than a ribonucleotide probe, no
RNase would be involved.  But even for ribo's, thionin works fine.  The
stain is light, but this is desirable, as otherwise the stain would obscure
the silver grains.  Am I missing something basic here?  Susan Bachus
----- Original Message -----
From: "J. A. Kiernan" 
To: "sanjay rakhade" 
Sent: Wednesday, March 26, 2003 2:04 AM
Subject: Counterstaining after in situ hybridization

> Grrr!
>   First.  You asked a question with a blank Subject line.
>   For this you deserve castration without anaesthesia etc.
>   That said, here's a possible answer to your question.
> The RNase used to remove molecules of your unbound probe also
> removes ribosomal RNA, which is what you were trying to stain
> with cresyl violet. That's why you can't do a Nissl stain - the
> Nissl substance (rRNA) has been removed.
> In my lab about 10 years ago we did some in situ with 35S on
> rats' medullas and encountered this problem, which we should have
> anticipated. The solution was simple: stain the basic proteins
> that accompany nucleic acids and are not attacked by nucleases.
> There was plenty of published guidance, with Lillie's bigger book
> (either of its last 2 editions) carrying plenty of references.
> We did this (Paragraphs quoted from the M&M in the MS of a
> paper):
> The slides were coated with Kodak NTB-2 nuclear track emulsion,
> and autoradiographs were prepared.  The exposure time was
> 22-days.  Some sections were counterstained with 0.1% fast green
> FCF at pH9.0 for 1h, washed, dehydrated, cleared and mounted in
> DPX, a resinous medium.  The counterstain was to show the shapes
> of the cells by virtue of their basic nucleoproteins [16], which
> had been unmasked by the RNase treatment.
> [Ref 16]
> 16 James J, Tas J. Histochemical Protein Staining Methods (Royal
> Microscopical Society Microscopy Handbooks 04). Oxford: Oxford
> University Press, 1984: 28-29
> We wrote this up, and your library should have the journal:
> Savedia, S. & Kiernan, J. A. 1994. Increased synthesis of
> ubiquitin mRNA in motor neurons after axotomy. Neuropathology and
> Applied Neurobiology 20, 577-586.
> The paper has more than 50 references, many of them relating to
> your question.
> (Sheila Savedia was the MSc student who did most of the lab work.
> She went on to qualify as a teacher and then got into a medical
> school. I don't know where she is now.)
> --
> -------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1
> _____________________________________________________________
> sanjay rakhade wrote:
> >
> > Hi everyone,
> >
> >             I am trying to set up a protocol for
> > counterstaining emulsion dipped slides (for in situ
> > hybridization using 35S labelled radioprobe) with cresyl violet
> > stain. I tried to do the same using 1% cresyl violet acetate
> > for 15 min followed by dehydration in serial 50, 70, 80, 95 and
> > 100% Ethanol and dipping in Xylene followed by Permamount. The
> > brain sections on my slide have the in situ grains intact,
> > however the cresyl violet stains the cells, especially the
> > neurons on the sections very lightly. I am not sure if this is
> > due to the emulsion , dehydration or some problem with my
> > staining protocol. I would appreciate it , if someone on this
> > list has tried this experiment before and can help me
> > troubleshoot ( or perhaps post a protocol for the same).
> >
> > Thanks in advance,
> >
> > Sanjay
> __________________________________________________________________________

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