Re: Mib-1 staining on wax sections of decalcified bone, adhesion prob lems after microwave antigen retrieval step
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | "Mendes, Maria" <mmendes@mtsinai.on.ca>, Histonet <Histonet@pathology.swmed.edu> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
Maria, Your problem sounds like one of how to mount sections
on slides rather than anything special to a particular tissue
or immunohistochemical method. You'll probably get lots of
helpful answers. Here are my suggestions, for what they're worth.
On Wed, 24 May 2000, Mendes, Maria wrote:
> problem. I would appreciate any help that could be sent my way. I may be
> contacted directly at mmendes@mtsinai.on.ca <mailto:mmendes@mtsinai.on.ca> .
Please don't ask for "private" replies. Questions and answers are of
interest to many histonetters.
[Sections coming off slides]
> The following procedures have been tried with no success thus far:
> 1) Contacted vendor that supplies Mib-1 to see if they had any
> information as well I did a search on Pubmed to find any articles on the
> subject
No luck with either? It's unlikely to be the primary antibody
that detaches the sections. A PubMed search for ADHESI* AND
(SECT* OR SLIDE*) ought to pull in lots of references.
> 2) Used positively charged slides from two different vendors
They are usually very good. You can also make your own, more cheaply,
using simple published methods (and with no threats of being sued
by the firms that sell such slides).
> 3) Used positively charged slides with albumin
This might neutralize the adhesive actions of the charged slide
surface and of either albumin or albumen used as a sticky protein
adhesive. Not recommended!
> filter paper rolled over the section to get any air out of the section
If there are air bubbles under sections, it means you're drying
them horizontally and too fast. The air un-dissolves from the
film of water between the floated-out section and the glass.
A later sentence in your email suggests that you may have heated
the mounted sections too soon. 37C comes before, not after melting
the wax.
> On all slides the wax was melted on the hot plate and the slides left
> overnight in a 37oC oven in some cases the slides were left over the
> weekend.
Let the slides stand to drain vertically for a few minutes (while
you're cutting or mounting some more sections), before putting
them on a hotplate. Also don't use a hot hotplate or other drying
device. It's disastrous if the wax melts while there's still some
water under the ribbon. A hotplate at 40C is fine for drying
drained slides. Heating to melt the wax is often a good
thing to do when the slides are fully dried (as many hours as
possible). Do it immediately before dewaxing. It can improve
flattening and speed up dissolving of the wax.
Mixing different adhesion-promoting methods is tempting ++ but it
isn't a good idea unless you have lots of time to do experiments.
Most sections, especially if thin (by which I mean 7 um or less),
adhere pretty well to untreated glass if the surface is clean.
Fierce treatments, especially hot liquids or alkaline reagents,
necessitate the use of slides that have been made extra-sticky.
Sadly, there are still boxes of "pre-cleaned" slides that are
dirty. I opened one a couple of weeks ago, and didn't need my
reading glasses to see that the slides were greasy and dusty.
Such slides must be washed and dried (with much cursing and
resentment) before using, with or without an adhesive.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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