Heike Grabsch wrote:

> - yes, I made my own Mayer's and filtered it after all chemicals were
> solved, however I do not filter it every day
>
> - I have already seen precipitates coming directly from haemalaun (or is
> it called haematoxylin? please forgive me for my ignorance), in my
> experience they have some sort of right angles and sharp borders and
> often are a little bit yellowish in the edge
>
> - may be,  I should describe my procedure again in more detail (like I
> did it today): so after IHC staining, I use AEC one step solution
> (BioGenex) as chromogen, rinse in dist. water, 90 sec Mayer's, first
> rinse in dist. water, then extensive rinsing with running cold (!) tap
> water, then alternatively 30 sec 0,5% Lithium carbonate or 0,25% ammonium
> hydroxide as suggested by Frieda this morning, thereafter extensive
> rinsing in running cold tap for may be 30 minutes, BECAUSE on both
> versions I had again these branched and spinous, deep blue precipitates
> and could not get rid of them. And of cause they are not all over the
> tissue section but mainly on top of the cells of interest! And you only
> see them on the tissue, no precipitate on the slide itself.
>
> As nobody else seem to have this problem, I must make a major mistake.
> So, from today's experiment I conclude it is probably not the lithium
> carbonate. So, may be it is something in our tap water? I will make my
> next trial tomorrow using only dist. water for rinsing. Let's hope for
> the best.
>
> any more ideas?

Perhaps the earlier proceedures are doing something that induces the ppt to
form?

1. Run the hematoxylin proceedure by itself, no IHC or AEC. Precipitate?
2. Run the AEC then the hematlxylin proceedure. Precipitate?
3. We already know what happens when all three are run. Precipitate!
4. Switch to very dilute (0.025%) Thionin or Toluidine Blue for a nuclear
counterstain.

Geoff
--
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
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