I would try longer rinses in water after the hematoxylin,
acid alcohol and ammonia water (running water would probably
be best).  Also I would go longer in the 95% alcohols that
are after the eosin.

Kim Merriam
TKT
Cambridge, MA



AliNeumann@aol.com wrote:

> Hi everyone, I am the director of a new small outpatient
> laboratory, and we have had a very persistent and
> recalcitrant problem with variability of staining (some
> sections light, others dark, especially on eosin but to a
> lesser degree hematoxylin).  We are soaking slides in
> xylene for 3 minutes in each of 3 bins with agitation,
> then staining them on a Varistain 24-4 with a regressive
> stain.  Our staining protocols have been:
>
> 1. 100% Alcohol 20 seconds
> 2. 100% Alcohol 10 seconds
> 3. 95% Alcohol    10 seconds
> 4. Tap Water 10 seconds
> 5. Hematoxylin 7 minutes
>     (Protocol Harris mercury free non-acidified type)
> 6. Tap Water 20 seconds
> 7. Acid Alcohol 2 seconds
> 8. Tap Water 20 seconds
> 9. Ammonia Water 2 minutes
> 10. Tap Water 10 seconds
> 11. 95% Alcohol 10 seconds
> 12. Eosin             15 seconds
> 13. 95% Alcohol 10 seconds
> 14. 95% Alcohol 10 seconds
> 15. 100% Alcohol 20 seconds
> 16. 100% Alcohol 20 seconds
>
>
> 1.  100% Ethanol 40 seconds
> 2.  100% Ethanol 40 seconds
> 3.  95% Ethanol 40 seconds
> 4.  Tap water               60 seconds
> 5.  Hematoxylin 10 minutes
>      ( Protocol Harris mercury free non-acidified  type)
> 6.  Tap water  (sink) 60 seconds
> 7.  Acid Alcohol 2 seconds
> 8.  Tap water  (sink) 60 seconds
> 9.  Ammonia water 2.5 minutes
> 10.  Tap water 5 minutes
> 11.  95% EtOH 1 minute
> 12.  Eosin 10  seconds
> 13.  95% EtOH 10 seconds
> 14.  95% EtOH 10 seconds
> 15.  100% EtOH 40 seconds
> 16.  100% EtOH 40 seconds
>
> We have had the same problem with both protocols, and
> process mostly small skin biopsies.
>
> Our acid and ammonia solutions are as follows:
>
> 1% acid alcohol
> 2000 ml of 70% EtOH
> 5 ml  of HCl (hydrochloric acid)
>
> 1% Ammonia water
> 1980 cc tap water
> 40 ml of ammonium hydroxide
>
> We have tried longer xylene pre-treatment with more
> agitation, and wiping off alcohol drops before removing
> the bin from the last alcohol, as well as various staining
> adjustments.  We process most of our tissue on a short 3
> hour cycle however believe this problem has persisted even
> on overnight-processed tissue.
> I would very much appreciate any advise any of you can
> give me on this problem.  Thanks very  much and have a
> happy holiday!  Alice Neumann M.D. Precision Pathology PC,
> Arvada CO  alineumann@aol.com  303-432-7855