| Animals (subjects) | | Sex: M | | Number of animals: not specified | | Age: not specified | | Mass: not specified | | Unit of mass: not specified | | Housing conditions: normal housing | | Annotation: Adult male albino rats of the Sprague-Dawley strain were used in all experiments. |
| | Experimental method | | Experiment type: immunohistochemistry | | Neuron/glia identification method: stain specific | | Staining frequency: 1:5 | 27, 7
Experimental details:
| Antigen: CRH
Antigen species: not specified
Source (producer): specified elsewhere
Primary antibody: rabbit antiserum
Primary antibody species: rabbit
Antibody type (monoclonal, polyclonal): polyclonal
Secondary antibody: goat-antirabbit
Secondary antibody species: goat
Immunoglobulin class: IgG
Control: staining pattern
Visualization method: autoradiogram
Visualization medium: slide
Annotation: Up to five one-in-five series of ...sections through the PVH were saved and then prepared for indirect immunofluorescence staining of cells that cross-react with antisera against CRF, neurotensin, oxytocin, or vasopressin using a conventional method (Sawchenko and Swanson, 1981) based upon the localization of a primary antiserum raised in rabbits using an affinity-purified, fluorescein-conjugated goat anti-rabbit IgG. To ensure the validity of our localization of CRFimmunoreactive neurons, three sera against CRF human alpha-globulin conjugates were used. Two of these (C24 and C30) are directed against the C- and N-terminal portions, respectively, of the ovine CRF molecule (Swanson et al., 1983; Vale et al., 1983). The third was prepared against a synthetic rat CRF (Rivier et al., 1983) conjugate. Each of these sera was used at a final dilution of 1:3000, and pre-incubation of each with 22 mg ml-l of the respective immunogen blocked specific staining of cells and fibers in the hypothalamus. All sera used for both normal immunohistochemistry and blocking experiments contained 10 mg/ml of the “carrier” protein used for immunization.
| | Antigen: oxytocin
Antigen species: N/A
Source (producer): Dr. F. Vandesande and Dr.
K. Dierickx (University of Ghent, Belgium)
Primary antibody: oxytocin antiserum
Primary antibody species: not specified
Antibody type (monoclonal, polyclonal): monoclonal
Secondary antibody: not specified
Secondary antibody species: not specified
Immunoglobulin class: not specified
Control: adsorption
Visualization method: autoradiogram
Visualization medium: slide
Annotation: Antisera against oxytocin and vasopressin were provided by Dr. F. Vandesande and Dr. K. Dierickx (University of Ghent, Belgium) and were cross-adsorbed in the solid phase against the heterologous antigen according to a procedure outlined elsewhere (Swaab and Pool, 1975). Specific staining with each antiserum was blocked when immunoprocessing was carried out using sera that had been pre-incubated with an excess (15 mg/ml) of the homologous synthetic peptide and then diluted to the working concentration of 1:2000.
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