200
BURT G. FEUERSTEIN et al.
Procedure
Case selection, sectioning, and staining
Tissue blocks are cut after a pathologic review of Hematoxylin and Eosin
(H&E) or other appropriately stained thin sections to verify diagnosis. Cut
three consecutive 5-J.lm sections from a formalin-fIxed paraffIn-embedded
block. Stain the middle slide with H&E and one or both of the rest with
methyl green. Mark the H&E slide to defIne the areas to be microdissected.
The experience and attention of the person who determines the region of
interest is critical.
Methyl green
staining of paraffin
sections
The sections are deparaffInized and stained with methyl green. This stain
is used because it does not interfere with amplifIcation or labeling reactions. Hematoxylin does interfere with these reactions. After staining, the
tissue can be stored for several weeks if necessary.
1. Xylene extraction x2, 3 min each.
2. 100% ethanol, 2 min.
3. 95% ethanol, 2 min.
4. 70% ethanol, 2 min.
5. 50% ethanol, 2 min.
6. distilled water, 2 min.
7. Dip 3-6 times or leave 15-60 s in 0.1-1% methyl green (Sigma, St. Louis,
MO). Start with the lower methyl green concentrations so the material
is not too heavily stained.
8. Rinse in distilled water.
9. Incubate in distilled water 1-5 min depending on staining intensity.
10. Air dry upright.
Microdissection and digestion
Reagents PCR/PK Buffer. lOx PCR buffer (l00 mM TrisHCI pH 7.5, 2 mM MgCh, 500
mM KCI, 1 mg/ml gelatin) supplied with the Taq polymerase kit (Boehringer/Mannheim). PCR/PK buffer is PCR buffer supplemented with 0.5%
Tween 20 and 0.5 mg/ml proteinase K (Life Technologies).
BURT G. FEUERSTEIN et al.
Procedure
Case selection, sectioning, and staining
Tissue blocks are cut after a pathologic review of Hematoxylin and Eosin
(H&E) or other appropriately stained thin sections to verify diagnosis. Cut
three consecutive 5-J.lm sections from a formalin-fIxed paraffIn-embedded
block. Stain the middle slide with H&E and one or both of the rest with
methyl green. Mark the H&E slide to defIne the areas to be microdissected.
The experience and attention of the person who determines the region of
interest is critical.
Methyl green
staining of paraffin
sections
The sections are deparaffInized and stained with methyl green. This stain
is used because it does not interfere with amplifIcation or labeling reactions. Hematoxylin does interfere with these reactions. After staining, the
tissue can be stored for several weeks if necessary.
1. Xylene extraction x2, 3 min each.
2. 100% ethanol, 2 min.
3. 95% ethanol, 2 min.
4. 70% ethanol, 2 min.
5. 50% ethanol, 2 min.
6. distilled water, 2 min.
7. Dip 3-6 times or leave 15-60 s in 0.1-1% methyl green (Sigma, St. Louis,
MO). Start with the lower methyl green concentrations so the material
is not too heavily stained.
8. Rinse in distilled water.
9. Incubate in distilled water 1-5 min depending on staining intensity.
10. Air dry upright.
Microdissection and digestion
Reagents PCR/PK Buffer. lOx PCR buffer (l00 mM TrisHCI pH 7.5, 2 mM MgCh, 500
mM KCI, 1 mg/ml gelatin) supplied with the Taq polymerase kit (Boehringer/Mannheim). PCR/PK buffer is PCR buffer supplemented with 0.5%
Tween 20 and 0.5 mg/ml proteinase K (Life Technologies).