[Histonet] Has anyone done ISH with fairly large FFPE sections?

[Patsy Ruegg]

We use a Tunnel kit from Chemicon and apply about 30ul of probe with a cs on
top, look into buying just the probe which is what goes first, for our
Chemicon kit we buy several vials of probe before we have to buy the entire
kit again.

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 Lewis,
Patrick
Sent: Friday, August 10, 2012 9:57 AM
To: 'Histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Has anyone done ISH with fairly large FFPE sections?

Hi Everyone,

I am going to do some ISH for EBER  on FFPE sections, and I have some
concerns on the logistics.

The kits I am looking at are for 50 rxns, and it seems like they expect to
use 20 uL probe per rxn.

My sections are rather large, covering about 2/3 of a standard slide.

How many uL will it take to make sure I get good coverage and have enough to
avoid drying out the slide?

I plan to cover slip them and use a humidity chamber, but even still, I am
worried that with such a small volume it don't be enough.

These kits are expensive, +$900.00 each and if I have to double/triple my
rxn volume that would use up the kit in a hurry.

I haven't fully familiarized myself with the protocol yet, so maybe my
concerns are unjustified, but if anyone has done some ISH, I'd appreciate
any advice you'd care to give.

Thanks

Patrick.

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