Re: "my" (his) problem ... (long)
|From:||Philip Oshel <firstname.lastname@example.org>|
John Kiernan's helpful reply reminded me of what I used to do with 50
- 100 micron gelatin-enrobed Golgi-filled brain sections. I cheated.
Collect the sections in an ice-cube tray -- one of the kind with lots
of little compartments floating on buffer.
For your purposes, stain as John mentioned.
From the buffer/stain/wash, etc. mount the sections on a slide,
*before* dehydrating, if you can.
Place a second slide on top of the sections, and paperclip the slides together.
Place the section/slide sandwiches in the series of dehydrating
agents on edge, allowing the agent to be drawn up between the slides
by capillary action.
Use bilbous paper or the like to pull the old reagent out the other
side, and help exchange the fluid.
Extend all your times in the dehydrating fluids, because of the
smaller volume used. I didn't have to add extra steps, though. I did
have to use 30 minutes or more in the fluids.
Continue this through the clearing agent (I used HistoClear, but this
applies to xylene, etc.).
Remove the second slide, drop on the mounting agent and place on the coverslip.
Spend two days cleaning up the mess.
It also helps to trim away as much gelatin as possible *before*
sectioning, and maybe some after sectioning.
>On Thu, 10 May 2001, Bonnie Wayne wrote:
>> Yes, the gelatin blocks are fixed in formaldehyde ...
>> ... hrs to several weeks. The blocks are fixed but not frozen.
>That must mean you cut the sections with a vibrating
>microtome (Vibratome). They should have excellent
>morphology, with little in the way of differential
>shrinkage spaces around small blood vessels and large
>neurons and, of course, no ice crystal holes.
>John A. Kiernan
>Department of Anatomy & Cell Biology
>The University of Western Ontario
>London, Canada N6A 5C1
Supervisor, AMFSC and BBPIC microscopy facilities
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison, WI 53706 - 1284
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)
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