[Histonet] air dry or heat dry
Maria.Mejia <@t> ucsf.edu
Mon Jul 12 17:55:33 CDT 2010
Let's see you are sectioning your (fixed) rat brain on the cryostat at 10ums. I'm assuming they are mounted on standard size superfrost
plus slides & since they are fixed sections & even if they were not fixed - after sectioning why not air-dry these sections standing up under
a fan for 2 hours or longer and store them in plastic slide boxes that hold either 5, 10, 15, or more slides. Only store enough slides
in each box for a particular IHC run. Since I don't know what you are staining for - I don't know if storing fixed cryostat sections at -80C is
necessary. This is your protocol.
Seal each plastic box with cryo tape & place inside a zip-loc bag - fold the bags to the size of box & hold in place with rubber band.
Place boxes inside a labeled 3-inch cryo box & store at -80C. When you are ready to run IHC, just remove only what you need
from the -80C freezer & quickly return the rest back inside -80C. Even though these sections are fixed (& for those not fixed), once your
remove slide boxes from -80C freezer - I would not open the box. Let them equilibriate at room temp on bench top for about 30 mins before
removing from plastic bag & slide box. Then run your IHC protocol.
Department of Neurosurgery
San Francisco, CA 94103
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jeffrey Thompson [JefThompson <@t> salud.unm.edu]
Sent: Friday, July 09, 2010 2:21 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] air dry or heat dry
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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