Using Organotypic Tissue Slices as Substrata for the Culture of Dissociated Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 238

placed onto Parafilm™ ( see Subheading 2.2.1. ). Holders ( see Subheading
2.2.3. ) are made to transfer disks with mounted tissue slices out of the
vibratome tray without damaging the slices.
The preparation and use of nitrocellulose disks as supports is somewhat
laborious ( see below and Subheading 3.2. ), and they are not suitable for trans-
mitted light microscopy. As an alternative, slices can be mounted onto com-
mercial culture well inserts. Several suppliers (Millipore, Bedford, MA, Costar,
Cambridge, MA, and Nunc, Wilsbaden-Biebrich, Germany) manufacture these
inserts with growth surfaces made from a variety of translucent or transparent
polymer materials (e.g., polycarbonate, cellulose, etc.) and treated with a vari-
ety of ECM molecules (e.g., collagen, laminin, etc.). Characteristics such as
autofluorescence, transparency, rigidity, and porosity vary greatly among the
different membranes and, based on the particular experimental needs, should
be taken into account when making a choice of insert to use.

1. Parafilm™ dishes: When pipeted onto nitrocellulose disks that are on Parafilm™

(American National Can), small volumes ( L) of solutions remain in a

bubble over the disk because of the hydrophobic properties of the Parafilm™.
Cut a circle of Parafilm™ (

≈ 100 µ

10-12 cm diameter), remove backing, and place
down in a 150-mm plastic Petri dish. Using both hands, hold the sheet flat and,
with the back edge of a forceps or other blunt metal object, scratch several short
lines, perpendicular to the edges of the Parafilm™, around its perimeter. This
will score the Parafilm™ into the plastic of the dish. Sterilize under ultraviolet
(UV) light (>1 h).

≈

2. Nitrocellulose disks: Sterilize the cutting edge of a cork borer (12 mm diameter)

with flame and/or ethanol. Place a sheet of sterile, black nitrocellulose (0.45 m;

Sartorius, Bohemia, NY) into a plastic Petri dish and press the borer into it to cut
out disks. Transfer the cut disks with a sterile forceps to another Petri dish for
storage.

µ

3. Holders for nitrocellulose disks: A holder is made from the cap of a 6-mL round

bottom tube (Falcon, #2063). The cap is inverted and used as a well, on the bot-
tom of which the tissue-bearing nitrocellulose disks will be placed. Take a plastic
cap and place it onto the cutting end of an 11-mm cork borer, as if to cap the end
of the cork borer as a tube. The indent at the top of the cap should just fit into the
hole of the borer. Using a large rubber stopper as a backing, punch a hole out of
the cap. This should leave the top of the cap with just a rim. Discard the rest of
the cap top that was cut out. Using a scalpel or razor blade, cut the length of the
cap in half, discarding the half that contains indents on the inner side of the cap
and saving the half that has the rim ( Fig. 3E ). Sterilize by storing in ethanol.

4. Concanavalin A: 1.3 mg/mL in DMEM (or other medium or balanced salt solu-

tion). Filter sterilize and store aliquots at -20

° C.

5. Complete medium: DMEM (glutamine free, 4.5 g/L glucose) supplemented with

10 µ g/mL transferrin, 5 mg/mL crystalline grade bovine serum albumin (BSA;