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" <>
To:"Mendes, Maria" <>, Histonet <>
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 <> .
 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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