Standardization of Immunohistochemistry and Receptor-NOS and i-NOS in the Cervix of Bitch with Pyometra
R. Volpato; M. Araujo; R. Laufer-Amorin; M. Lopes; C. Ackermann
Nitric oxide is a modulator of physiological and pathological processes in mammals and appears to interfere with the neck relaxation process, its increase elevates the activity of metalloproteinase, apoptosis and glycosaminoglycan synthesis, alterations associated with cervical ripening process. Through immunohistochemistry we compare groups of pyometra closed and open cervix understanding better the involvement of NOS in the cervical opening mechanism, but it was necessary to standardize this tissue in dogs. Antigen retrieval was in a solution of 10 mM sodium citrate incubation in a 750 W microwave for 10 minutes. Then blocks were performed in a solution of 8% H2O2 for 20 minutes, Molico® 3 g/100 ml for 60 minutes and protein block for 30 minutes. The primary antibody incubation Rb pAb to e-NOS and i-NOS was from Abcam® at 1:100 dilution in a humid chamber for 2 hours at 37°C. The material was then incubated with the secondary antibody for 30 minutes Histofine® and development with DAB chromogen for 5 minutes. Slides were counterstained with Mayer's hematoxylin for 1 minute. We then performed with dehydration in alcohol baths and assembly of the blades. The cell cytoplasm stained blue is negative and stained brown are considered positive, thus showing the efficiency of immunohistochemical technique to assess receptor-NOS and i-NOS in the cervix of bitches.