[Histonet] Cryostat sections with small bubbles
abright <@t> brightinstruments.com
Thu Jun 16 05:11:38 CDT 2005
Basically you need to section brain at -8 to -12ºC as colder temperatures will cause what you are getting plus cracking. However to achieve good quality sections at these temperatures you will need to maintain the knife and anti-roll temperatures at -20ºC or lower to allow the section to slide in between the knife and the anti-roll plate unimpeded. Which is what Kathleen is doing when she warms the block face with her finger, a technique that went out with the Ark and should not be used as firstly you could have an infection issue and secondly it is not a very scientific approach in this day and age when there are devices fitted to some cryostats such as independent specimen temperature control to perform this task with full and repeatable control and of most importance, ease.
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From: Kathleen Spencer [mailto:kspencer <@t> utmem.edu]
Sent: 15 June 2005 16:50
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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