[Histonet] RE: Eosin too pink

From:Gayle Callis

Paul made good suggestions here. Remember that carryover of eosin into the 
first 95% alcohol rinse can become excessive, so rotating or changing the 
95% alcohol stations should be done more often (we sometimes do this daily 
with large volume staining sessions) to ensure proper differentiation or 
removal of excess eosin per his suggestion and as it was taught to me 
milleniums ago).  If there is excessive carryover of eosin in the 95%, 
staining may continue with this dye - resulting is overly red tissues.

If there is any pink or red tinge in the 100% alcohol or last station 
before clearing not only is dye being moved up the dehydration chain of 
events but also water (see Richard Allan website , go to their product 
Staining Guide.)

  At 01:04 PM 1/13/2006, you wrote:
>         You can try rinsing the slides in tap water after staining, as
>someone already suggested.  However , the pH of tap water differs
>considerably from one locality to another.  If your tap water is slightly
>acidic it won't differentiate eosin at all. If it is very slightly basic (pH
>7.1-7.2) you may be able to use it to remove excess eosin at a more or less
>controlled rate, and will have to experiment to determine the optimum time
>of exposure.  But if your tap water is more strongly basic it may remove
>eosin so quickly that the differentiation is difficult to control, and
>uneven differentiation is likely to occur.  I would feel safer using 95%
>ethanol to partially extract eosin from overstained tissues.  Eosin
>dissolves out of tissue fairly quickly in 95% ethanol, but very slowly in
>100% ethanol.  In any such differentiation procedure the rack of slides
>should be frequently agitated in the solvent to ensure evenness of
>extraction throughout the section.
>         That having been said, if I were in your position I wouldn't think
>in terms of correcting the overstaining, but preventing it.  An extremely
>short staining time is likely to cause uneven staining (remember, the eosin
>has to extract the previous solvent, whether water or alcohol, from the
>tissue before it can get into the tissue and stain it).  I would try
>diluting the eosin, using whatever solvent it is made with, until the
>desired intensity of staining can be achieved in a normal time period.
>Histonet mailing list

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

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