[Histonet] in situ with paraffin sections

[Tony Henwood]

Emily,

Unfortunately, there are a lot of superfluous steps involved in ISH. It
reminds me of the initial IPX protocols. There were many unfathomable
steps included that seemed to incite a feeling of magic required to
obtain results. Simplifying the technique was one of the main reasons
why routine labs were able to utilise it. I hope the same will occur
with ISH.

IPX and ISH are similar:

Block the endogenous enzyme
Reverse the formalin cross-linking
Apply the probe or antibody
Demonstrate the bound probe or antibody.

Stringency washes might be required if there is a high possibility of
cross-hybridisation.

Tissue sections will react differently to Molecular biology nucleic acid
containing tubes and gels
Granted, for DNA hybridisations you will need to denature the DNA strand
but overall that is about it.

I would keep it simple and go from there.
Many of the added steps were added by researchers because they seemed
like the right thing to do not because they were required. 

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Emily
Sours
Sent: Thursday, 20 August 2009 2:49 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] in situ with paraffin sections


Hello

I've been reading a few protocols for RNA in situ hybridization with
paraffin sections.  One of the suggestions is to dehydrate the sections
on the first day after rehydration and pre-hybridization processing
(para fix, proteinase K, acetylation, pbs washes between all).  This
seems counter intuitive to me, or at least unnecessary.  Any suggestions
why this would be done? Our protocol does not involve this, by the way,
and it works fine except for the tissue tearing.


Emily

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