[Histonet] problem with brain sections falling off subbed slides
one_angel_secret <@t> yahoo.com
Thu Nov 3 07:46:48 CDT 2011
Good morning. I see you mention only part of your protocol so I am not sure if you are going directly to the stain? This procedure seems close to what you are working with, it has a few tips. Hope this helps.
1. Mount frozen or vibratome sections on gelatin coated or positive charged plus slides. Air dry sections or bake slides on slide warmer overnight.
2. Place slides directly into 1:1 alcohol/chloroform overnight and then rehydrate through 100% and 95 % alcohol to distilled water. DON’T put frozen sections directly to water otherwise they will come off the slides.This is called de-fat step that will reduce background fat staining.
3. Stain in 0.1% cresyl violet solution for 5-10 minutes. Notes:Staining in warmed cresyl violet solution (warm up in 37-50 ºC oven) can improve penetration and enhancing even staining. It is particularly beneficial for thicker (30 um) sections.
4. Rinse quickly in distilled water.
5. Differentiate in 95% ethyl alcohol for 2-30 minutes and check microscopically for best result.
6. Dehydrate in 100% alcohol 2x5 min.
7. Clear in xylene 2x5 min.
8. Mount with permanent mountingmedium.
From: John Kiernan <jkiernan <@t> uwo.ca>
To: Douglas M Burns <dmburns9 <@t> gmail.com>; histonet <@t> lists.utsouthwestern.edu
Sent: Wednesday, November 2, 2011 11:19 PM
Subject: Re: [Histonet] problem with brain sections falling off subbed slides
Gelatin alone is not an adhesive because it dissolves in water. Chrome alum-gelatin is an excellent adhesive for sections and costs almost nothing to make. Subbed slides are one coated with chrome gelatin, dried and stored. The following web link shows a 1999 publication giving the rationale and methods for a variety of section adhesives.
= = =
On 02/11/11, Douglas M Burns <dmburns9 <@t> gmail.com> wrote:
> I am new to working with brain, and I am having a bad problem with
> both thin and thick rat brain sections sliding off of subbed slides.
> We tried commercial treated slides first, but then moved on to
> subbing them. The subbing was done with gelatin according to a printed
> protocol, and I can see the film on the dried slide. When kept horizontal
> they seem okay, but as soon as they are put into a slide holder for washing
> or anything, they begin to slide off or even move around. After a short
> while, many/most of mysteriously vanished (into the wash solution).
> Our slices are sliced by cryostat from a rat brain that has been
> fixed with 4% paraformaldehyde and 30% sucrose. The slice comes right off
> the cryostat, and then I m ove it onto a warm slide. (Perhaps they are not
> warm enough?? I have seen others blow on them to warm them.) Slides
> destined for Cresyl violet staining were dried for 3 days at room
> temperature before being moved.
> It is the Cresyl violet protocol that causes the most trouble, but I
> am seeing some problems with any type of large volume wash. At this point,
> I don't have enough experience with this specific tissue and our protocols
> to be able to tell for sure what is happening. Thus, I am entirely open to
> advice - any advice that might help with the problem.
> Thank you ------------- Doug Burns, Kansas City
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