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

[McMahon, Loralee A]

We buy rubber cement from walmart or staples and seal the outside of the coverslip before the hybridization step.  And we have had much success with minimal probe.   
But I understand, I cross my fingers when I do large resected pieces of liver.  AND I keep on complaining to the vendors that they do not provide enough probe to cover paraffin sections of large size.   

Loralee McMahon, HTL (ASCP)
Immunohistochemistry Supervisor
Strong Memorial Hospital
Department of Surgical Pathology
(585) 275-7210
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lewis, Patrick [patrick.lewis <@t> seattlechildrens.org]
Sent: Friday, August 10, 2012 11:57 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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