Re: [Histonet] sections falling off

From:"Susan Bachus"

I know that  poly-L-lysined slides are supposed to work for this, but I have 
NEVER seen sx's fall off good old-fashioned double-gelatin-subbed slides 
under any conditions!  Might be worth a try.   Susan
----- Original Message ----- 
From: "Olek Michalski" 
To: "Histonet" 
Sent: Wednesday, February 06, 2008 12:08 PM
Subject: Re: [Histonet] sections falling off

Well, what can I say? It seemed a little silly to me too but I was told
this step is for removing fat (myelin) which does not allow a good
contrast in Nissl staining. Nevetheless most of us use chlorofom/ethanol
mixture instead of xylene as a first step and I have never seen such a
strange "ruffling" on sections processed this way even if sections fall
off sometimes.
It is common here to cut strongly fixed tissue. It is collected in PBS
filled wells or on gelatin covered slides. In the latter case the sections
are spread using brush and drop of buffer and air-dried (no alcohol stage
between cutting and drying). Maybe it would be better to keep them in
higher temperature for some time as suggested Mary? I am afraid that the
problem is in too strong fixation. Any comments are wellcome.

Olek Michalski

2008-02-05 19:30:54 Rene J Buesa  wrote:

> If they were cryosectioned she did not have to go through xylene or=20
> graded alcohols to "dewax and hydrate" since there was no dehydration or 
> wax infiltration to begin with.
>  Cryosectioned sections just need to wash away the medium used to=20
> cryosection (OCT perhaps?) and just stain with the aqueous solutions.
>  Later they can de dehydrated and cleared if a permanent mount is=20
> desired.
>   Try to float the sections in a water bath and pick them up and don't 
> try again to use xylene or alcohols.
>   Probably she was following a procedure for paraffin embedded tissue 
> that should have been adapted to frozen sections and it was not.
>   René J.
> Olek Michalski  wrote:
> Dear Histonetters,
> a friend of mine just faced a massive sections falling off. She is doing
> Nissl staining in mouse brain (brains perfused with formalin, postfixed 3
> days, and cryosectioned) on poly-L-lysined slides. She just managed to
> pass trough xylene and decreasing grades of alcohol to water and the
> sections started to detach. We were trying to reattach them but it went
> out that sections are not flat any more. It seems like the tissue is
> rehydrated unevenly, but keeping it in water for hours didn't work. Is
> there any way to spread these sections not damaging them at the same 
> time?
> She is likely to rescue this sections even if some work is needed.
> Best regards
> Olek Michalski

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