[Histonet] Cryostat sections with small bubbles
BDUE <@t> PARTNERS.ORG
Wed Jun 15 11:53:50 CDT 2005
Hello Emily, I have two or three tricks you might try, and a comment. The
"bubbles" are probably similar to what I get whe picking up 10um human muscle.
The tissue "jumps" to the slide in a semi-random fashion no matter how carefully
you arrange the pick-up. So the "bubbles" are actually regions that get
encircled before flattening out fully on the slide. They are really wrinkles
that get "trapped" because the tissue can't spread once it hits the slide.
Imagine trying to toss a wet blanket so it lands perfectly flat.
1.) try picking up on cold slides and then warming the back of the slide with
your finger and air dry for a few minutes. If your sections are cold and flat,
you may be able to get them to lay flat on a cold slide. The cold slide won't
"grab" the section like a warm slide. But you do need to warm everything up to
get proper adhesion. You can handle thick FS with forceps, especially if there
is an OCT border. By controlling how fast you warm the slide, you can to some
extent control how the section flattens.
2.) perfused tissue seems ok for you, so fixation is ok. Try floating your 20um
fresh frozen sections on a small cup of cold PFA or cold formalin. You could put
the fixative bath in your cryostat. Let them float flat like on a paraffin water
bath. Pick up on charged or coated slides and air dry. Be careful about
floaters. Don't float too long or the fixative will crosslink over the charged
sites which you need for adhesion. (This is part of the reason that your
perfused sections don't stick well.)
3.) Bancroft (5ed p. 103) lists a great section adhesive that I have used with
thick fixed frozens. It should work with your 20um perfused floating sections. I
use it modified, as follows. Make A) 1% gelatin in warm water and B) 2%
formaldehyde (I use 37% formaldehyde, but you could probably adjust for PFA or
NBF). Combine A and B 50:50 shortly before use. The gelatin slowly crosslinks
and the soln is only usable for a few hours at room temp. To use, wet the
surface of a charged slide with the soln -- use a swab or sponge or something.
You only need a film of the adhesive, not a dripping wet slide. You pick up your
FS on the wet slide. This takes a little practice to keep the crystat stage from
getting covered. The sections spread flat on the film of adhesive. Then air dry
the slide for 5-20 min. until visibly well dry -- a 37C oven is ok too. The
sections will not fall off. I have done 20um fixed frozens of rat spinal cord
(incl. cauda equina) and done silver stains and H&Es and LFBs, and not a single
fiber ever fell off. The only down side is that the gelatin around the sections
will pick up stains.
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From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Kathleen
Sent: Wednesday, June 15, 2005 11:50 AM
To: Katri Tuomala
Cc: histonet <@t> lists.utsouthwestern.edu; Emily Jane Wiesner-Camm
Subject: Re: [Histonet] Cryostat sections with small bubbles
I cut rat brain sections at -18 to -20 and often have to warm the block
face with my finger before each section. I also have better luck if I
cut sections at 10u. These are fresh frozen brains. If I cut them at
20u I get bubbles under the sections. I keep them in the cryostat, and
then fix the sections in cold fix, buffer wash, water wash, then air
dry them in the hood.
When I cut rat brain that has been perfused, I cut 20u floating
sections. They never adhere well to slides, even if I use a hot plate.
I always have bubbles under the section. That is why in this case we
use floating sections.
I hope this is helpful.
On Jun 14, 2005, at 8:39 PM, Katri Tuomala wrote:
> Hi Emily,
> I have never heard of hot plating cryostat sections. You may be
> boiling the moisture under the section forming bubbles or do you air
> dry them well first? Also -35 C seems too cold for brain sections, you
> may be getting some cutting artifacts.
> I'm sure there'll be others with helpful information for you.
> Katri Tuomala
> Hamilton, Ontario, Canada
> ----- Original Message ----- From: "Emily Jane Wiesner-Camm"
> <Emily.Wiesner <@t> medecine.unige.ch>
> To: <histonet <@t> lists.utsouthwestern.edu>
> Sent: Tuesday, June 14, 2005 10:36 AM
> Subject: [Histonet] Cryostat sections with small bubbles
>> Hi All,
>> I was wondering whether anyone can let me know why small bubbles
>> appear underneath my rat brain sections when I cut them on a cryostat
>> (at -35) when they appear perfectly flat before placing them on a
>> hotplate. How they can be avoided?
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
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