[Histonet] air dry or heat dry
Rene J Buesa
rjbuesa <@t> yahoo.com
Sat Jul 10 11:52:28 CDT 2010
The "rule of thumb" is that you never freeze-thaw-freeze protein containing substance (a tissue section is in this category as well as any protein containing solution).
Freezing-thawing-freezing can end denaturing the proteins and your IHC results could be compromised.
You can safely take out of the box just the slides you need and let them thaw at room temperature.
So, answering your specific question, the second group is doing what I used to do and, consequently, I think it is the best way (no surprises there!).
--- On Fri, 7/9/10, Jeffrey Thompson <JefThompson <@t> salud.unm.edu> wrote:
From: Jeffrey Thompson <JefThompson <@t> salud.unm.edu>
Subject: [Histonet] air dry or heat dry
To: histonet <@t> lists.utsouthwestern.edu
Date: Friday, July 9, 2010, 5:21 PM
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.
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