Materials

1-mm3 (or larger) fresh tissue specimen

PBS (APPENDIX 2A)

Culture medium (optional)

50 to 400 ^g/ml pepsin in 0.01 M HCl (from porcine stomach mucosa, 2500 to

3500 U/mg protein, Sigma; store dilution up to several weeks at 4°C) 1% formaldehyde in PBS (freshly prepared from a 37% formaldehyde stock) 70%, 90%, and 100% ethanol

90-mm petri dishes 100-^m nylon filter

Poly-L-lysine- or organosilane-coated slides (see Support Protocol) Shandon Cytospin cytocentrifuge (Life Sciences) or equivalent Coplin jars

Additional reagents and equipment for cell counting (appendix 3a)

1. Place a fresh 1-mm3 (or larger) tissue specimen in a 90-mm petri dish containing 1 ml PBS or culture medium. Scrape and cut with a razor blade for a few minutes to disaggregate the cells. Filter the suspension through a 100-^m nylon filter to remove large aggregates.

The filtered cell suspension contains both intact cells and isolated nuclei from ruptured cells.

At this stage, the nuclear suspension can be fixed in 70% ethanol (-20°C) or 3:1 methanol/acetic acid and stored (appendix 3B) for up to several years at -20° to -30°C without reduction of ISH reactivity. When the number of nuclei is small, fixation in ethanol is preferable.

2. Count cells and nuclei in freshly prepared or ethanol-fixed cell suspension. Prepare a suspension of ~5 x 105 cells and nuclei/ml PBS.

3. Spot 5- to 10-^l aliquots of suspension onto coated slides either by dropping several microliters directly onto the slides or by cytocentrifuging 200 ^l PBS containing 5 to 10 ^l suspension in a Shandon Cytospin cytocentrifuge 5 min at 75 to 100 x g, room temperature (also see units 5.2 & 8.2).

Concentration of cells and nuclei will vary with size of specimen, and aliquot volume will vary with concentration and with the cell density required on the slide. To obtain maximum nuclear recovery, do not cytocentrifuge small (<50 ul) or large (>200 ul) volumes.

In general, cytocentrifugation will lead to a more equal distribution of cells.

4. Air dry 15 min at room temperature.

If necessary, heat slides 30 to 60 min at 80° C to obtain adequate cell adhesion.

5. Incubate slides 20 min at 37°C in 100 ml of 50 to 400 ^g/ml pepsin in a Coplin jar.

Optimal conditions should be determined empirically. In general, 100 jug/ml is a standard pepsin concentration. For mild permeabilization, slides can be immersed in 0.1 M HCl containing 0.05% Triton X-100 or Tween 20 for 15 min.

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