Metadata

Experiment acronym Annotation Collator Author checked
Esclapez-GAD65/67In situ hybridization study of GAD67 and GAD65 expression in the rat brain.Mihail Botano

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: GAD67 cDNA 2.7 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 GAD67 cDNA (2.7 kb) was a subclone of the GAD67 clone isolated from a hgt-11 rat whole brain library (Tillakaratne et al., '92). 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.

Anatomy and histology
Section plane: coronalAngle: not specifiedCutting method: microtome Preservation: freezing Thickness: 30 micrometer
Staining type: none Sampling: 1:2 Annotation: Thirty micrometers thick brain sections were cut on a cryostat and collected in 0.01 M phosphate-buffered saline, pH 7.4 (1 x PBS). Adjacent sections were processed with rat GAD67 and GAD65 probes, using nonradioactive in situ hybridization methods.

Mapping details
Coordinates: noneData presentation:
text
images of all labeled sections
Mapping approach: brain region is captured in a BAMS nomenclature other than that used in the original publication