[Histonet] opinion on heating slides prior to IHC

[Rene J Buesa]

I have done that many times and from my experience on the issue this us what I did and recommend you to do:
1- having slides at -80ºC is OK but what is not OK is to warm them before taking the sldies out to re-freeze them again because you should never freeze-thaw-freeze any protein.
2- you take your box with the slides out of the freezer, remove the slides you are going to use and return the box with the rest of the slides to the freezer as quick as you can
3- thaw the slides to room temperature and after that heat them to help fixing the sections to the slides
4- that will not "cook" the epitopes that have already been fixed with the para formaldehyde. The epitopes have been already cross-linked and will not be denatured when heating the sections
5- the sections, as you describe, are FS from para formaldehyde perfused brain so they have NOT been dehydrated, so why do you need to "re-hydrate" what has not been dehydrated in the first place?
From your description the "other camp" is closer to the best procedure.
René J.


________________________________
From: Jeffrey Thompson <jefthompson <@t> salud.unm.edu>
To: histonet <@t> lists.utsouthwestern.edu 
Sent: Monday, August 6, 2012 2:59 PM
Subject: [Histonet] opinion on heating slides prior to IHC

Dear Histonetters,

We have an ongoing debate in our lab regarding preparing tissues for staining. I would like to get input from the wider community to use in our discussions.

In all cases the tissues in question were from paraformaldehyde perfused rat brains that were sectioned at 10 or 20 microns on a cryostat and affixed to Superfrost Plus slides and stored in slide boxes within ziplock bags with dessicant at -80 C. 

So the debate is what happens when preparing to stain:

One camp thinks that the slide box/bag should be warmed to 50 C before removing the slides to drive off any water from condensation on the tissues. Following this they will remove the slides to be stained and return the box to the freezer. After applying the PAP pen border then proceed with the staining protocol with serial alcohol rehydration as the first step.

The other camp feels that heating the slides may potentially 'cook' the epitopes as well as being redundant since the first incubation in 100% EtOH will drive off any water which will be immediately added back in by the procedure anyway. They favor removing the frozen slides from the box and immediately returning the box with remaining slides to the freezer, and air drying the slides to be stained at RT before applying the PAP pen border

The epitopes of interest are various intracellular and transmembrane cell type markers and enzymes common in rat neural tissue.

Any input will be appreciated

Thanks,

J Thompson

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