| Animals (subjects) | | Sex: M | | Number of animals: 14 | | Age: not specified | | Mass: 150-250 | | Unit of mass: | | Housing conditions: not specified | | Annotation: The tissue for this study was obtained from 14 adult male Sprague-Dawley rats (150-250 g). |
| | Experimental method | | Experiment type: in situ hybridization | | Neuron/glia identification method: morphology | | Staining frequency: not specified |
Experimental details:
| Measured nucleic acid: mRNA
Source (producer): not specified
Probe sequence: GAD65 cDNA 2.4 kb
Sequence species: not specified
Probe sequence orientation: antisense
Control: sense
Labelling method: antigen labelling
Visualization method: colorimetry
Visualization medium: slide
Annotation: The antisense (complementary to cellular mRNA) and control sense (identical to cellular mRNA) rat GAD67 and GAD65 probes used in this study were digoxigenin-labeled riboprobes of approximatively 160 nucleotides in length. These RNA probes were produced by in vitro transcription of two previously described GAD cDNAs. The rat GAD65 cDNA (2.4 kb) was isolated from a lambdazapII rat hippocampus library (Erlander et al., '91). GAD67 and GAD65 cDNAs, each containing the entire coding region, were subcloned into the Bluescript transcription vector (SK polylinker, Stratagene Cloning Systems, La Jolla, CA) in both orientations in order to obtain antisense and sense riboprobes. The lengths of time in the chromogen solution were
determined according to two different protocols. In one series of experiments, sections processed for each of the GAD mRNAs were incubated for identical lengths of time in the chromogen solution in order to compare the staining directly. In other experiments, the sections were incubated in the chromogen solution until optimal staining was achieved for each GAD mRNA. Optimal staining was defined as a maximum number of specifically stained neurons (maximum sensitivity) with a low background of
general tissue staining and no nonspecific staining of cell bodies.
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