Differential immunolabeling for electron microscopy of diverse peptidergic neurons.

Abstract

We describe a simple and reliable method for differential immunolabeling of pre- and post-synaptic signal peptides at the ultrastructural level. Hypothalamic tissues of rats, including the suprachiasmatic nucleus, were cut on a Vibratome. Visualization of the immunolabeling of somatostatin (SRIH) and vasoactive intestinal polypeptide (VIP) was performed with avidin-biotin-peroxidase-diaminobenzidine (DAB). The end product of the DAB to VIP was further silver-intensified in a physical processing using silver nitrate, and the silver grains were finally substituted for gold. DAB-labeled SRIH fibers synapse on gold-labeled VIP perikarya and dendrites in the suprachiasmatic nucleus.

Photic regulation of peptides located in the ventrolateral subdivision of the suprachiasmatic nucleus of the rat: daily variations of vasoactive intestinal polypeptide, gastrin-releasing peptide, and neuropeptide Y

“…These observations suggest that VIP-L1 neurons receive various inputs and project to both subdivisions of the SCN, while GRP-LI neurons receive restricted inputs and project mainly to the ventrolateral SCN. Indeed, from electron microscopic studies, VIP-L1 neurons in the SCN receive several kinds of inputs from neurons immunoreactive for SS (Maegawa et al, 1987), 5-HT (Hisano et al, 1988a), and NPY (Hisano et al, 1988b). Thus, it could be speculated that light suppressed VIP-L1 levels in the SCN because visual information was integrated on the VIP neurons with several inhibitory signals, such as SS (Koch and Schonbrunn, 1984), NPY (Shibata and Moore, 1988), and 5-HT (Meijer and Groos, 1988).…”

Substance P-Containing Neurons Innervating LHRH-Containing Neurons in the Septo-Preoptic Area of Rats

“…Animals were anesthetized with an intraperitoneal injection o f sodium pentobarbital (40-50 m g/kg body weight) and perfused via the ascending aorta with 0.1 M phosphate buffer (PB) (pH 7.4) followed by a fixative consisted of 4% paraformal dehyde, 0.1% glutaraldehyde and 0.2% picric acid in 320 mosm PB(pH 7.4). The hypothalami were dissected out and immersed in the same fixative for 1 2 -18 h at 4 ° C. Frontal sections were cut at 30-40 pm on a Vibratome (Oxford instrument, USA) and processed for the double labeling procedure [17], Sections were first treated with 0.2% Triton X-100 in phosphate-buffered saline (PBS) (pH 7.4) for I h at room temperature, and then the following procedures were carried out: (1) incubation in anti-SP serum diluted 1:2.000 for 3 h at 18 °C; (2) biotin ylated goat anti-rabbit IgG (Vector Laboratory, Burlingame, Calif., di luted 1:100) for 1.5 h at 18 °C ; (3) avidin-biotin peroxidase complex (ABC, Vector, mixing 45 pi of both avidin and biotin peroxidase in 5 ml of 0 .1 M PB, pH 7.4) (4) visualization with the solution consisted of 1.5 mg of 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 20 pi of 3% hydrogen peroxide in 20 ml PBS: this gave a brown immunoreaction product for SP; (5) incubation in anti-LHRH serum diluted 1:1,000 or in anti-gonadotrophic hormone-releasing hormone-asso ciated peptide (GAP) serum diluted 1:5,000 for 12-18 h at 18 °C; (6) biotinylated goat anti-rabbit IgG diluted 1:100 for 12-14 h at 18 0 C: (7) silver-gold intensification of the DAB chromogen for SP [17]; (8) ABC for 12-14 h at 18 °C ; (9) visualization o f the immunoreaction for LHRH or GAP with DAB chromogen. The sections were mounted on glass slides coated with albumin, sealed with balsam, and examined histologically.…”

Use of ultrastructural immunohistochemistry in human pituitary pathology

“…In the combined pre-and pre-embedding method, both the first and second labelings are done on the coronal sections cut at 30 to 60 p. m thickness on a Vibratome. Two antigens are distinguished at the electron microscopic level by gold or ferritin particles and DAB reaction products, respectively (Leranth et al, 1985;Maegawa et al, 1987;Van den Pol et al, 1985), and DAB reaction products and tetramethylbenzidine (TMB) reaction products, respectively (Norgren and Lehman, 1989). Both DAB and TMB are peroxidase-mediated products, but the ultrastructural appearance of TMB reaction products is visibly different from that of DAB (Norgren and Lehman, 1989).…”

“…As for the application of double labeling on the same section (Table 3), the post-embedding method using colloidal gold particles of different sizes appears superior to other methods, chiefly because of its simple methodology and applicability to the routinely processed resin-embedded tissues (Bendayan, 1982). Double labeling procedures by the pre-embedding method (Maegawa et al, 1987) or by the combination of the preand post-embedding methods (Vila-Porcile et al, 1987) have been developed, but they seem more complicated technically than the colloidal gold post-embedding double labeling method.…”