Skin biopsies (4 mm) from each site and normal skin were taken from the rodents. Biopsy tissue was fixed in 10% buffered formalin (Fisher Scientific, Pittsburgh, PA, USA) and processed for permanent paraffin embedding on an ASP 300 tissue processor (Leica Microsystems, Bannockburn, IL, USA). Approximately 6 µm thick paraffin sections were cut and collected.
The paraffin sections were deparaffinized according to routine procedures. The antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at 90°C for 12 hours. The sections were then incubated in a humidified chamber overnight at 4°C with a pP70S6K antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:50 dilution). A biotinylated anti-mouse secondary antibody was incubated with sections for 2 hours at room temperature after the primary antibodies’ reaction. An indirect biotin avidin diaminobenzidine (DAB) system (Dako, Glostrup, Denmark) was used for detection.
The paraffin sections were deparaffinized according to routine procedures. The antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at 90°C for 12 hours. The sections were then incubated in a humidified chamber overnight at 4°C with a pP70S6K antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, 1:50 dilution). A biotinylated anti-mouse secondary antibody was incubated with sections for 2 hours at room temperature after the primary antibodies’ reaction. An indirect biotin avidin diaminobenzidine (DAB) system (Dako, Glostrup, Denmark) was used for detection.