Re: [Histonet] tumor cryosection

From:"Olek Michalski"

Dnia 28-11-2006 o 16:05:07 Adi Sabag   
napisał:

Indeed, I take the slide out of the cryostat. I don't need the tissue to =20
be frozen after cut. If you do, you could try sucrose solution but I have  
no idea weather it would help.
I use a separate brush to transfer liquid onto section. Just wet the brush  
and cover the tissue with small volume of buffer. When tissue soaks it  
detaches from the glass slide, so be aware. I consider it an advantage  
since I can adjust the section (it sometimes folds or sticks incorrectly).

On the other hand I remember the notion about removing air bubbles with  
dry brush just after collection on the slide. Try searching the list  
archive (couple of months ago there was an extensive discussion on  
cryosectioning technique).

Last thought: I do not flash freeze my tissue after soaking with sucrose =20
solution. I work with brains, also fixed w/ 4% PFA. I soak tissue with  
sucrose in two step procedure: first 15% sucrose until the tissue sinks  
and then in 30% sucrose again until sinks. Then I embed it in OCT and  
freeze in -20 or on dry ice (large specimens). I think it's posible that =20
your section thaw inequally (ie the part which doesn't come into contact =20
w/ glass keeps more rigid) and that's your problem.

Best regards
Olek Michalski

> Dear Olek,
> Thank you for your reply.
> I have some questions;
> After I pick up the section I keep the slide inside the cryostat. Do you  
> do that
> also or do you leave it outside? Because insidee the cryostat the PBS  
> will
> freeze and than I don't see how it can help...
> Do you put the drop of PBS on the tissue or on a region in the slide  
> without
> tissue that is adjacent and then the PBS soaks toward the tissue?
>
> Thank you!
>
> Adi Sabag.
>
>
> Quoting Olek Michalski :
>
>> Dnia 27-11-2006 o 09:25:01 Adi Sabag 
>> napisa┬│:
>>
>> > I'm working with s.c tumors that are approximately 8x8 mm, they were
>> > fixed with
>> > 4% PFA and then either frozen in isopentene over liquid N2 or I used a
>> > protocol
>> > of embedding in sucrose and then freezing. In both cases I found that  
>> the
>> > optimal temperature for getting nice sections is around 20C. I'm  
>> using 3
>> > different sizes of sections- 60, 30 and 10um. The problem is that when
>>
>> Well, after picking a section I put a drop of PBS onto the slide and let
>> the tissue soak. Then it tends to spread on the glass and it's even
>> possible to remove an air bubble from underneath with fine brush (while
>> working w/ 40um - thinner e. g. 20um tend to tear too much). Of course I
>> have to wait till tisue dries to stick to the glass again.
>>
>> Hope this helps
>> Olek Michalski
>>
>
>




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