[Histonet] sections falling off in day 2 ISH (Megha Kumar)

Patsy Ruegg pruegg <@t> ihctech.net
Tue Aug 14 12:27:22 CDT 2012

I would use good charged slides like VWR Superfrost Plus or Fisher + slides
and try to cut the sections thinner, sections thicker than 4-6 microns fall
off easier.  It is also important to airdry slides completely before baking
them, we bake at 60dc for an hour to overnight in an oven after airdrying.
For ish we do it manually on a hot plate and put a glass coverslip over the
section to keep it from drying out during high temp long incubations
required for ISH. 

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting, LLC
40864 Arkansas Ave
Bennett, CO 80102
Phone: 303-644-4538
Fax: 720-859-4110
pruegg <@t> ihctech.net
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Therese C.
Sent: Friday, August 10, 2012 12:05 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] sections falling off in day 2 ISH (Megha Kumar)

Hi Megha, Not sure why your sections are falling off. When I did ISH I  used
Colorfrost Plus Slides. I would cut at 4 microns-mouse or human.  I would
dry them at room temp a few hours followed by 55 C for an hour. I would use
the slides the next day or next week, never the same day. On day 2 my SSC
was 60 C and I used a stir bar for 2 hours on slow stirring. Rarely did I
lose any sections.
Terri Thinnes
Research Assistant
The Scripps Research Institute
La Jolla, CA

Message: 13
Date: Fri, 10 Aug 2012 14:40:30 +0530
From: Megha Kumar <meghak <@t> g.clemson.edu>
Subject: [Histonet] sections falling off in day 2 ISH
To: histonet <@t> lists.utsouthwestern.edu
	<CAGnJi_VJCmLodt3F1zqdk7B79XN3nZCD7UKDigBOanbdybG8Gw <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

Hello everyone!
When I subject murine skin and intestine paraffin sections (7-10microns) to
ISH protocol, the sections fall off on day 2 when I add the SSC. The
sections are taken on ploy lysine coated slides and remain on the slides
when I do other protocols such as antibody staining. Can anyone suggest me
why this is happening? How to prevent the slides from falling off on day 2?
Thank you for all the help!


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