Dab reagent kit

The tissues were excised and immersed in 4% paraformaldehyde (PFA). After fixation, the tissues were decalcified in 10% sodium citrate and 22.5% formic acid for 6 weeks at 4°C. Staining was performed on 6 μm paraffin-embedded sagittal (mesio-distally) sections. After deparaffinization, the slides were incubated with Proteinase K (10 μg/mL, AM2546, Thermo Fisher Scientific, United States) for 20 min at 37°C. Subsequently, the slides were incubated with antibodies against collagen IV (1:500 dilution, ab6586, Abcam, United Kingdom), fibronectin (1:400 dilution, ab2413, Abcam, United Kingdom), plakophilin (1:100 dilution, ab230855, Abcam, United Kingdom), or laminin-5 (1:200 dilution, ab14509, Abcam, United Kingdom) at 4°C overnight. The specimens were sequentially incubated with secondary antibodies and streptavidin peroxidase. The results were visualized following staining with a diaminobenzidine (DAB) reagent kit (Invitrogen, United States). The sections were counterstained with Mayer’s hematoxylin. All the specimens were observed using a stereomicroscope (MD5500D; Leica, camera: DFC495; Leica, Lens: HCX PL APO 409; Leica).

The tissues were excised and immersed in 4% paraformaldehyde (PFA). After fixation, the tissues were decalcified in 10% sodium citrate and 22.5% formic acid for 6 weeks at 4 °C. Staining was performed on 6 μm paraffin-embedded sections. After deparaffinization, the slides were incubated with Proteinase K (10 μg/mL, AM2546, Thermo Scientific, USA) for 20 minutes at 37 °C or, for GFP, with pepsin (Digest-All™ 00–3009, Invitrogen, USA) for 10 minutes at 37 °C. Subsequently, the slides were incubated with antibodies against periostin (1:1,000 diluted, ab14041, Abcam plc, UK), fibrillin1 (1:500 diluted, ab53067, Abcam plc, UK), vWF (1:100 diluted, AB7356, EMD Millipore Co., USA), HLA (1:100 diluted, ab70328, Abcam plc, UK), nestin (1:200 diluted, MAB353, Millipore co. USA), panCK (1:50 diluted, MS-343-P0, Thermo Scientific, USA), or GFP (1:500 diluted, 2955 S, Cell Signalling Technology, USA) at 4 °C overnight. The specimens were sequentially incubated with secondary antibodies and streptavidin peroxidase. The results were visualized following staining with a diaminobenzidine (DAB) reagent kit (Invitrogen, USA). The sections were counterstained with Mayer’s haematoxylin. All specimens were observed using a stereomicroscope (MD5500D; Leica, camera: DFC495; Leica, Lens: HCX PL APO 409; Leica).