Anti cgrp antibody

We fixed lungs in formalin for 24 h before paraffin embedding. Paraffin blocks were sectioned at four-microns and stained with hematoxylin and eosin. Paraffin sections were cut and processed from xylene through a graded ethanol series (100%, 95% and 70%) to PBS. Unmasking was performed using microwave heating in sodium citrate buffer (0.01M at pH6.0). Endogenous peroxidases were blocked with 3.5% H₂O₂, and immunohistochemistry was performed with an overnight incubation with anti-CGRP antibody (Sigma C8198, 1:1000). Biotin-conjugated secondary antibodies (Vector Laboratories) were used at a dilution of 1/200 in blocking solution. After secondary antibody binding, detection was performed via a biotin-peroxidase complex (Vectastain ABC, Vector Laboratories) with DAB substrate (Vector Laboratories). Haematoxylin was used to counterstain.

Two and a half hours or four hours after the SIF or IS administration, the rats were perfused transcardially with 50 ml phosphate-buffered saline (PBS, 0.1 M, pH 7.4), followed by 200 ml 4% paraformaldehyde in phosphate buffer under chloral hydrate anesthesia, and the trigemino-cervical complex was removed and postfixed overnight for immunohistochemistry in the same fixative. After cryoprotection, 30 μm cryostat sections were cut and serially collected in wells containing cold PBS. The free-floating sections were rinsed in PBS and immersed in 0.3% H₂O₂ in methanol (CGRP staining) or PBS (nNOS and TRPV1 staining) for 30 min. After several rinses in PBS containing 1% Triton X-100, sections were kept overnight at room temperature in anti- CGRP antibody (Sigma, C8198) at a dilution of 1:20000, or TRPV1 antibody (Santa Cruz, s.c.28759) at a dilution of 1:1000, or for two nights at 4 °C in anti-nNOS antibody (EuroProxima, 2263B220–1) at a dilution of 1:5000. The immunohistochemical reaction was visualized by the Vectastain Avidin-Biotin kit of Vector Laboratories (PK6101), and nickel ammonium sulfate-intensified 3,3′-diaminobenzidine. Control experiments included the omission of the primary antisera.