Prb 165p

The tissue immunofluorescence staining method has been described previously (Lee et al., 2016 (link)). Antibodies of H3 K4me3 (1:1,000, no. 39159, Active Motif), H3 K9me3 (1:2,000, ab8898, Abcam), H3 K27me3 (1:2,000, no. 07-449, Millipore), CD34 (1:200, no. 553731, BD Biosciences), mouse α-K14 (1:100, ab7800, Abcam), Ki67 (1:500, ab15580, Abcam), AE13 (1:50, IQ292, ImmuQuest), BMP4 (1:200, ab39973, Abcam), K1 (1:200, PRB165P, Covance), K10 (1:100, PRB159P, Covance), CD31 (1:200, no. 550274, BD Biosciences), Tenascin (1:500, AB19013, Millipore), pSmad1 (S463/465), 5 (S463/465), 9 (S465/467) (1:100, 13820S Cell Signal), and Vimentin (1:1,000, a gift from Dr. Yan Lammerding, Cornell University) were used.

5-µm-thick mouse or human tissue sections were incubated with 1x Blockace (DS Pharma BioMedical) in PBS-T for 30 min, washed three times with PBS-T for 5 min each, and incubated with rabbit anti–mouse or –human PLA2G2F antibody (Degousee et al., 2002 (link)) at 1:500–1,000 dilution in a 10-fold-diluted Blockace for overnight at 4°C. The sections were then washed 3 times with PBS-T for 5 min each time and incubated with Alexa Fluor 647–labeled goat anti–rabbit IgG antibody (Molecular Probes; 1:1,000) at 20°C for 1 h. For double immunostaining, the sections were washed three times with PBS-T for 5 min each and incubated with rabbit antibodies against mouse loricrin, cytokeratin 1 and cytokeratin 5 (PRB-145P, PRB-165P and PRB-160P [Covance], respectively; 1:500) prelabeled with Alexa Fluor 555 (Zenon Labeling System; Molecular Probes) at 20°C for 1 h. Counterstaining was performed with 4,6-diamino-2-phenylindole (DAPI; Vector Laboratories). Stained sections were analyzed with a confocal laser-scanning microscope (LSM510 META, Carl Zeiss). Human skin sections were obtained by surgery at Chiba University (Chiba, Japan) after approval by the Faculty ethics committee and informed consents from patients.