Using Organotypic Tissue Slices as Substrata for the Culture of Dissociated Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 236

Fig. 3. Steps to making cocultures of dissociated cells plated onto living tissue
slices. The tissue to be sliced is dissected (A) , embedded in agarose and sliced on a
vibratome (B) , and then placed onto nitrocellulose disks for support (C) . Disks with
mounted slices are transferred from the vibratome using a holder made from the cap of
a round-bottom tube ( D , see Subheading 2.2.3. ). A hole is punched in the top of
the cap, leaving a rim, and the cap is shortened (E) so that the disk can be placed in the
bottom of the cap, forming a well (F) . After transfer from the vibratome, disks are
removed from the holder ( see Subheadings 3.2.4. and 3.2.5. for details), and placed
either on parafilm under a bubble of medium or in tissue culture wells (G) . To obtain
a cell suspension, the tissue is dissected (H) , dissociated (I) , and labeled with a fluo-
rescent vital dye (J) . Cells are then allowed to settle onto the slice cultures (K) .
those in the slice. Cells can be similarly labeled with membrane-intercalating
vital dyes such as DiI (3) . Bisbenzamide, a blue-fluorescent, nuclear stain, is
useful for making visible the overall anatomy of cultured slices. Protocols for
immunostaining such slices can be found elsewhere (1 , 2) .
We optimized this method for making living slices of perinatal mouse fore-
brain to use in acute (<24 h) cocultures with dissociated primary neurons. The
sectioning protocol should be applicable for use with most tissues, but may