[Histonet] problem with brain sections falling off subbed slides

[Kim Donadio]

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. 
 
Procedure:
	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.
 
http://www.ihcworld.com/_protocols/special_stains/nissl-frozen-section.htm
 
Kim Donadio


________________________________
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.
     http://publish.uwo.ca/~jkiernan/adhesivs.htm

John Kiernan 
Anatomy,UWO 
London, Canada
= = = 
On 02/11/11, Douglas M Burns <dmburns9 <@t> gmail.com> wrote:

> 
> Hello,
> 
>       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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> 
> 


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