Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations

Abstract

A method is described for the immunohistochemical localization of peptides in whole-mount preparations. Tissue is fixed as laminae with a picric acid/formaldehyde mixture and then dehydrated, cleared and rehydrated before exposure to antibodies. This procedure ensures adequate penetration of the antibody molecules without the need to freeze and thaw the tissue or to use detergents, preserves antigenicity and lowers non-specific background staining. The laminae are incubated with the primary antisera for 16 h at room temperature and, after washing, with a second, fluorescent tagged, antiserum. This can be followed by a peroxidase-anti-peroxidase localization of the second antiserum, which acts as a bridge. The method gives a precise and reproducible localization of immunoreactive peptides, with good penetration and low background even in thick preparation. Large areas can be scanned and neuroeffector relationships studied more easily than in sections.

Nitric oxide, and not vasoactive intestinal peptide, as the main neurotransmitter of vagally induced relaxation of the guinea pig stomach

“…Male Hartley guinea pigs (300-350 g) (Costa et al, 1980) For cross-sections of the stomach wall, the stomach fundus was fixed as before and washed in PBS containing 15% sucrose and 0.01 % sodium azide. Cryostat blocks were prepared from small pieces of tissue (2 x 1 cm) and 7-81tm sections were cut at -20°C, transferred to poly-L-lysinecoated slides and allowed to dry for 1 h. They were immunostained for NOS using the indirect avidin-biotin complex (ABC) immunoperoxidase method (Polak, 1988 (Graham & Karnovsky, 1976).…”

Myenteric enkephalin neurons around the laser-photocoagulation necrosis: An immunocytochemical investigation in the guinea pig jejunum and proximal colon.

“…The immunocytochemical techniques which we used in the present study were apparently more sensitive than the previous method (COSTA et al, 1980a). However, it was still impossible to demonstrate the weakest immunoreactivity in the thin enkephalin neuron processes.…”

“…We immunostained whole-mount preparations of perfusion-fixed intestinal tissues with the recently improved cytochemical procedures (KoBAYASHI et al, 1984(KoBAYASHI et al, , 1985. Preservation of the immunoreactivity and the fine structure of the enkephalin neurons seemed better than in the samples prepared by the previous methods (COSTA et al, 1980a). Therefore, we could visualize a series of regrowth structures of the enkephalin neurons around the laserphotocoagulation necrosis (laser necrosis) in detail.…”

Distribution of Argininosuccinate Synthetase-like Immunoreactive Neurons in the Rat Myenteric Plexus: A Whole Mount Study

“…Samples of the esophagus, stomach, and small and large intestines were immediately removed and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.2% picric acid for 2 days at 4°C. After fixation, samples were subjected to the whole-mount preparation technique of Costa et al (1980). For immunocytochemistry, whole-mounts were preincubated in 1% goat serum and 0.3% bovine albumin in PBS, and then incubated for 7 days at 4°C with rabbit antiserum to ASS (IgG concentration:…”