Hcx pl apo 409

Samples were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and then embedded in paraffin using standard procedures. Serial paraffin sections (4-μm thickness) were prepared for Hematoxylin and Eosin (HE), immunostaining and in situ hybridization. Antigen retrieval was achieved by citrate buffer, pH 6.0. After antigen retrieval, immunohistochemical analyses were performed using the DakoCytomation Envision System (using horseradish peroxidase with diaminobenzidine enhancer) (Dako, United States) according to the manufacturer’s instructions. The slides were incubated with antibodies against Pan-cytokeratin (Pan-CK) (1:50, Thermo Fisher Scientific^TM, United States), FGF10 (1:50, Santa Cruz, Unite States), Caspase 3 (1:1600, Cell Signaling Technology, United States) and Ki67 (1:200, Abcam, United Kingdom). The specimens were sequentially incubated with secondary antibody and streptavidin peroxidase. Finally, the results were visualized following staining using a diaminobenzidine reagent kit (Invitrogen, United States). The sections were counterstained with hematoxylin. All specimens were observed by stereomicroscope (MD5500D; Leica, camera: DFC495; Leica, Lens: HCX PL APO 409; Leica). At least 10 mice were examined in each experiment.

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).

After the CT scan, maxillae were hemisected, and each tissue was immersed in 4% paraformaldehyde for up to 24 h. 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 pepsin (Digest-All^TM 00–3009, Invitrogen, United States) for 10 min at 37°C. Subsequently, the slides were incubated with antibodies against MMP-9 (1:100 diluted, AB19016, EMD Millipore Co., United States), myeloperoxidase (MPO, 1:200 diluted, Rb-373-A0, Thermo Fisher Scientific, United States) or CD3 (1:100 diluted, ab5690, Abcam plc, 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 specimens were observed using a stereomicroscope (MD5500D; Leica, camera: DFC495; Leica, Lens: HCX PL APO 409; Leica).