[Histonet] air dry or heat dry

[andreahooper <@t> rocketmail.com]

I recommend to just take out the slides you need one at a time. 

If you are concerned about icy slides take them out rapidly without removing the box from the freezer (ie: do it on the shelf in the -80 deg). Also add some dessicant to the slide box. If its a major concern, use smaller slide boxes, or cut less slides, or individually heat seal each slide or small set of slides into their own plastic envelopes.

I cut the OCT slides. Dry for about 4h then put at -80 deg C. After freezing I do not allow the slides to warm at all. They go directly from the slide box in freezer into the PFA (or PBS depending). Great morphology.

I do not recommend you heat your sections once, much less the whole box repeatedly. This seems like a bad idea over time for sure. Especially if you are trying to repeat experiments and conditions.

Why do you acetone fix when your tissues are already PFA fixed? This seems unnecessary.

If I had to choose from your selection the best technique, I say the air dry group in the hood for 30 min is best.

Andrea

Sent from my Verizon Wireless BlackBerry

-----Original Message-----
From: "Jeffrey Thompson" <JefThompson <@t> salud.unm.edu>
Sender: histonet-bounces <@t> lists.utsouthwestern.edu
Date: Fri, 09 Jul 2010 15:21:47 
To: <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] air dry or heat dry

Hello,
 
We have an ongoing debate in our lab regarding the relative virtue of heat drying slides vs RT air drying them.
 
Tissues: 4% paraformaldehyde perfused rat brains frozen in OCT blocks
 
Sections: 10 microns on a cryostat (at approx -25 C)
 
Slides: Superfrost Plus
 
The slides are stored in slide boxes at -80 C until staining.
 
When staining the heat dry faction (wanting to avoid icy slides) put their slide boxes straight out of the freezeer into a 50 C oven for 30 minutes before taking out slides to stain. then the box goes back to the freezer until the next round of staining, 
 
The air dry group feels that cooking the antigens repeatedly at 50 C is problematic so they take the frosty slides out their slide boxes and return them ASAP to the freezer. The slides to be stained are air dried in the fume hood for about 30 min.
 
A third, middle of the road, person takes her slides out of the cold boxes and then puts the slides in a 60 C oven for 30 minutes.
 
For all three groups then, the slides are given a PAP pen border to prepare for IHC and when the pen solution is dry, 10 minutes in acetone and the remainder of the staining procedure.
 
So my question is: Who is using the best technique? 
 
Another hotly debated topic is wether it is advisable to put a drop of PBS on the slide before 'sticking' the section to prevent folds in the sections..
 
Any opinions are appreciated.
 
Thanks,
 
Jeff   
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