Immunofluorescence Analysis of NF-κB and iNOS

Immunofluorescence was realized according to the previously described methodology of Guerra et al. (2016) (link) and Ribeiro et al. (2017) (link). Sections of the oral mucosa from the hamsters, three per group, were dewaxed using xylol and, posteriorly, washed in concentrations different of ethanol and PBS. In process of antigen retrieval, was utilized a 10 mM sodium citrate solution containing 0.05% Tween 20 for a period 40 min at 95°C. Posteriorly, the samples were incubated in Sudan-Black 0.1% diluted in 70% ethanol, for 40 min, at room temperature to decrease the tissue autofluorescence. The slides again were incubated at 4°C overnight with mouse monoclonal anti-NFκB (1:200; sc-8008, Santa Cruz Biotechnology) or rat polyclonal anti-iNOS (1:200, sc-8332, Santa Cruz Biotechnology) primary antibodies and were washed three times for 5 min in PBS containing 0.2% Triton X-100. Subsequently, the samples were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500 in 1% BSA blocking solution; Dako, Brazil, and United States). Finally, coverslips were applied using VECTASHIELD^® mounting medium with DAPI (4′,6-diamidino-2′-phenylindole). Immunofluorescence images were obtained using a Carl Zeiss Laser Scanning microscope (LSM 710, 20× objective, Carl Zeiss, Jena, Germany) (Araujo Junior et al., 2016 (link); de Assis et al., 2016 (link)).