4. Transfer tissue samples into the automatic tissue processor (see
Note 10) to dehydrate by exposing the sample to ethanol
solutions with increasing ethanol concentration. After dehydration in ethanol, infiltrate in liquid paraffin. Immerse the
samples in the following solutions:
(a) 75% ethanol, 30 min.
(b) 80% ethanol, 30 min.
(c) 85% ethanol, 30 min.
(d) 90% ethanol, 30 min.
(e) 95% ethanol, 45 min.
(f) 100% ethanol, 45 min.
(g) Isopropanol, 45 min.
(h) Isopropanol, 45 min.
(i) Roticlear, 45 min.
(j) Roticlear, 45 min.
(k) Paraffin, 6 h.
(l) Paraffin, 24 h.
5. Transfer tissue samples infiltrated with paraffin into embedding
templates, and pour more hot paraffin wax (60
C) over the
samples. Cool the paraffin blocks on a cold plate (at À10
C).
6. Make serial paraffin sections of 10 μm thickness using a manual
rotary microtome. Transfer sections onto glass slides.
7. Dry and flatten specimens on a heated plate (40
C) for several
hours.
8. In order to remove paraffin from the sections, first incubate the
slides at 60
C for 30 min. To rehydrate the specimens, subject
the slides to the following incubations at room temperature:
(a) Xylene, 10 min.
(b) Xylene, 10 min.
(c) 100% ethanol, 2 min.
(d) 90% ethanol, 2 min.
(e) 80% ethanol, 2 min.
(f) 70% ethanol, 2 min.
(g) Distilled water, 0.5 min.
9. Stain the slides with hematoxylin-eosin staining solution, to
visualize nuclei and the general tissue architecture (see
Note 11).
(a) Incubate slides in hematoxylin solution for 8 min, followed by 10 min washing under running tap water.
(b) Incubate slides for 5 min in eosin solution.
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Willem F. Wolkers and Andres Hilfiker
Note 10) to dehydrate by exposing the sample to ethanol
solutions with increasing ethanol concentration. After dehydration in ethanol, infiltrate in liquid paraffin. Immerse the
samples in the following solutions:
(a) 75% ethanol, 30 min.
(b) 80% ethanol, 30 min.
(c) 85% ethanol, 30 min.
(d) 90% ethanol, 30 min.
(e) 95% ethanol, 45 min.
(f) 100% ethanol, 45 min.
(g) Isopropanol, 45 min.
(h) Isopropanol, 45 min.
(i) Roticlear, 45 min.
(j) Roticlear, 45 min.
(k) Paraffin, 6 h.
(l) Paraffin, 24 h.
5. Transfer tissue samples infiltrated with paraffin into embedding
templates, and pour more hot paraffin wax (60
C) over the
samples. Cool the paraffin blocks on a cold plate (at À10
C).
6. Make serial paraffin sections of 10 μm thickness using a manual
rotary microtome. Transfer sections onto glass slides.
7. Dry and flatten specimens on a heated plate (40
C) for several
hours.
8. In order to remove paraffin from the sections, first incubate the
slides at 60
C for 30 min. To rehydrate the specimens, subject
the slides to the following incubations at room temperature:
(a) Xylene, 10 min.
(b) Xylene, 10 min.
(c) 100% ethanol, 2 min.
(d) 90% ethanol, 2 min.
(e) 80% ethanol, 2 min.
(f) 70% ethanol, 2 min.
(g) Distilled water, 0.5 min.
9. Stain the slides with hematoxylin-eosin staining solution, to
visualize nuclei and the general tissue architecture (see
Note 11).
(a) Incubate slides in hematoxylin solution for 8 min, followed by 10 min washing under running tap water.
(b) Incubate slides for 5 min in eosin solution.
736
Willem F. Wolkers and Andres Hilfiker