Biology Reference
In-Depth Information
require some modifications depending upon the properties of the particular
tissue used. Similarly, the dissociation protocol was optimized for neural tis-
sue and may require modification for other tissues or for particular experimen-
tal requirements.
We found that, after 1 d in culture, the tissue slices may begin to flatten.
This process alters anatomical structure and borders. The particular questions
and needs of the investigator will determine whether longer culture periods are
needed and if such in vitro alterations could affect the interpretation of data.
Regardless of the culture period, investigators should take care to ensure that
particular results are not because of artifacts such as folds, tears, or other
incongruities in the tissue slice. A wise approach is to examine all fixed,
mounted slices for such defects prior to examining the behaviors of the labeled
dissociated cells plated on them.
2. Materials
All solutions should be sterile (except paraformaldehyde). Unless noted, all
chemicals are from Sigma, St. Louis, MO. All media and balanced salt solu-
tions are from Gibco, Gaithersburg, MD. Procedures should be performed asep-
tically and, when possible, in a laminar flow hood.
2.1. Embedding and Slicing Tissue
1.
Phosphate Buffered Saline (PBS): 137 m
M
NaCl, 2.68 m
M
KCl, 7.83 m
M
Na
2
HPO
4
, 1.47 m
M
KH
2
PO
4.
2.
PBS/glucose: PBS supplemented with 0.45% glucose and 1 m
M
sodium pyru-
vate (from 100X stock, Gibco). Filter sterilize.
3.
Low melting point (LMP) agarose solution: 2% LMP agarose (Gibco-BRL) in
PBS. Autoclave to dissolve agarose and to sterilize. When cool, but still liquid
(<50
°
C), supplement to 0.3 % final glucose with sterile 30% glucose stock. Stable
at 4
°
C in sealed bottle at least 6 mo.
4.
Vibratome tray (Technical Products International, Inc., St. Louis, MO) and teflon
coated vibratome blades (Ted Pella).
5.
Tissue adhesive, such as Locktite™ (Ted Pella) or any comparable glue may work.
2.2. Mounting Slices onto Supports for Culture
We chose to mount tissue slices onto black nitrocellulose disks because this
material provided the rigidity we needed for our experimental manipulations.
Black was chosen to minimizes reflection and scattering of light during exami-
nation by fluorescence microscopy. To ensure that slices attach well to the
support, the disks are pretreated with the lectin concanavalin A, however, other
proteins (e.g., laminin, fibronectin, etc.) may work as well or better for a par-
ticular tissue. For this treatment and for coculturing, disks can be placed into
12- or 24-well flat bottom tissue culture plates. Alternatively, disks can be
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