Using Organotypic Tissue Slices as Substrata for the Culture of Dissociated Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 242

already on the slices or after removing a portion of it ( see Note 25 ). Coculture in
a humidified atmosphere at 37 ° C ( see Note 15 ).

2. To fix cocultures, slowly pipet warm paraformaldehyde/sucrose under the

medium of the cocultures. Alternatively, you can transfer disks directly to warm
paraformaldehyde ( see Note 26 ). Fix at room temperature for 15-30 min. Stop
the fix by replacing it with PBS ( see Note 27 ). Fixed cocultures can be stored at
4 ° C in PBS with 0.02% sodium azide.

3. To fluorescently counterstain the nuclei of cells in the slice, add bisbenzamide

(10 g/mL final in PBS) to live or fixed cocultures at room temperature and

incubate for at least 10 min ( see Note 28 ).

µ

4. To mount cultures on microscope slides, a “stage” is built up on the slide to hold

the coverslip over the coculture without crushing it. Use superglue to affix two
22 22 mm coverslips on a slide at least 15 mm apart but so that a third coverslip

can bridge across them when placed over the gap. Place a disk on the slide in the
space between the two stages and fill the area with mounting medium ( see Note 29 ).
Overlay the third coverslip and aspirate off any excess solution. Seal the edges
with nail polish and let dry. Fixed and mounted cocultures can be stored at 4

×

° C.

4. Notes
1. The tissue may sink to the bottom of the dish. To prevent this, pour the agarose in
two layers. Pour a bottom layer, and when it begins gelling, but is not yet solidi-
fied, pour a top layer and add the tissue. It should sink to the top of the solidifying
bottom layer.
2. Any space created by liquid trapped between the tissue surface and the hardened
agarose can cause uneven slicing. To prevent this, thoroughly coat the tissue by
moving it around in molten agarose before it hardens. Avoid making bubbles.
3. To minimize the deflection of the block each time the blade enters it, make cuts
to taper the side of the block that the blade will enter. When placed in the tray, the
taper of the block should point toward the blade like an arrow.
4. If the glue does not hold well, there may have been too much liquid on the agar-
ose. Aspirate excess liquid before placing the block on the glue. Dry glue on the
tray can be removed with a razor blade.
5. Oil placed on the blades by the manufacturer can be removed with ethanol. If
flame-sterilizing the blades, minimize exposure to heat since this can dull and
distort the blade edge.
6. At this thickness, slices of embryonic and postnatal mouse forebrain were sturdy
enough to be manipulated and not tear, but were thin enough so that flattening
and distortions of the slice surface were minimal during culture. Some tissues
may allow thinner, or require thicker, sectioning.
7. For embryonic and postnatal mouse forebrain, a relatively slow speed and the
maximum vibration provided the best settings to begin cutting the tissue. After
most of the slice had been cut, however, the vibration intensity was lowered
to minimize damage to the delicate, sectioned tissue. In general, the softer the
tissue, the slower the speed and the greater the vibration needed.