Immunohistochemical Staining of Skin Sections

Immunohistochemical staining of skin sections with specific antibodies [phospho-p44/42 MAPK (ERK1/2) (#4370, Cell Signaling Technology; 1:400), phospho-mTOR (#2976, Cell Signaling Technology; 1:100) and Ki-67 (Thermo Fisher Scientific; prediluted)] was performed on a Ventana Discovery XT automated IHC research slide staining system (Roche, Tucson, USA). For antigen unmasking, deparaffinized slides were pretreated with Cell Conditioning Solution (Ki-67) or citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) (phospho-ERK1/2 and phospho-mTOR). Ki-67 proliferation index was determined as described previously (Almeida et al., 2012 (link)).