Cdk4 c 22

Mouse tissues were washed, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned. Feet tissue was additionally decalcified with 15% EDTA pH 7.2. Primary antibodies used for immunohistochemical stainings were CDK4 (C-22) from Santa Cruz, p-S6 S240/244 (rabbit polyclonal) and cleaved caspase 3 (5A1E) were purchased from CST. Ki-67 (H-300) was from Santa Cruz or from Abcam (ab66155), Mac-2 (M3/38) was obtained from Cederlane and MPO was from Dako. Biotinylated horse anti-mouse IgG or biotinylated horse anti-rabbit IgG were from Vector Laboratories. Novocastra streptavidin-HRP (Leica) and AEC-high sensitivity substrate chromogen (Dako) were used for detection of primary antibodies. Alternatively VECTASTAIN Elite ABC Kit and DAB (Vector Laboratories) were utilized. For immunofluorescent staining of paraffin slides, primary antibodies used were p-S6 S240/244 from CST, Mac-2 (M3/38) either from Cederlane or directly labelled from Biolegend and F4/80 (CI:A3-1) from Serotec. Species-matched secondary antibodies (A488 and A546) were purchased from Invitrogen. Some images were quantitatively analyzed with ImageJ. Percentage of positive staining was determined by calculating the ratio between stained area and total tissue area within a predefined threshold. All pixels in the image with values below the threshold were counted.