H & E Stain
(hematoxylin and eosin)
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Harris hematoxylin* is NOT recommended. Gill's and Mayer's formulations are
suitable.
Frozen sections should be placed in 70% ethanol for 30 - 60 sec. prior to staining.
DO NOT allow them to dry or thaw prior to being put in the ethanol.
*One of the constituents of Harris hematoxylin mimics nucleotides and will interfere with
PCR reactions.
1. Sterile distilled or RNase-free water 10 sec.
2. Gill's or Mayer's hematoxylin 10 sec.
3. Sterile distilled or RNase-free water 10 sec.
4. Bluing reagent 15 sec.
5. 70% ethanol 15 sec.
6. Eosin Y in 70% ethanol 15 sec.
7. 95% ethanol 15 sec.
8. 95% ethanol 15 sec.
9. 100% ethanol 15 sec.
10. 100% ethanol 1 min.
11. Xylene 5 min. Slides can be held in xylene until you need them.
12. Air dry on a Kimwipe in the fume hood for 5 min. Slides can be kept in a
box with desiccant while setting up the scope.
13. Proceed immediately to LCM.
Rapid staining and dehydration are very important in maintaining the integrity of the
DNA/RNA/proteins in your samples, especially in frozen tissue.
Staining should be performed as close as possible to the scheduled LCM
transfer time using solution baths that are replaced regularly. Staining
should be performed as follows:
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1. 70% ethanol fix
2. purified water wash
3. Mayer's hematoxylin (1-2 min.)
4. purified water wash
5. Bluing reagent (30-60 sec)
6. 70% ethanol wash
7. 95% ethanol wash
8. Eosin Y (1-2 min.)
9. 95% ethanol wash (x2)
10. 100% ethanol wash
11. xylene wash for 1 min
12. air dry for at least 2 min. to allow the xylene to evaporate
completely.