Immunohistochemistry Standardization of FAS and FAS-L Receptors in the Cervix of Bitch with Pyometra
R. Volpato; M. Araujo; R. Laufer-Amorin; M. Lopes; C. Ackermann
The families of proteins that regulate apoptosis include cell death receptors such as FAS-L cytokine that interacts with FAS receptor complex forming FAS-L/FAS. Cytokines and apoptosis are factors related to cervical ripening and may be involved in the cervical opening mechanism in bitches with pyometra open and closed cervix. To compare the two groups immunohistochemistry can be used to understand the involvement of FAS and FAS-L in this mechanism, but it was necessary to standardize this tissue in dogs. Antigen retrieval was in citrate solution with 10 mM sodium incubation in pressure cooker Pascal®. 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. Incubation the primary antibody Santa Cruz Biotechnology® FAS and FAS-L at 1:500 dilution was in a humid chamber for 2 hours at 37°C. The material was then incubated with the secondary antibody for 30 minutes Histofine® and developed with DAB chromogen for 5 minutes. Slides were counterstained with Mayer's hematoxylin for 1 minute. We then performed dehydration in alcohol baths and assembly of the blades. The cell cytoplasm stained brown are considered positive and blue is negative, thus showing the efficiency of the immunohistochemical technique for evaluation of FAS and FAS-L receptors in the cervix of bitches.