Counterstaining after in situ hybridization

From:"J. A. Kiernan"

  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

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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