Mki67

The femurs and cranial base from Osx-cre, Osx-Fgfr3 and Col1-Fgfr3 mice and their control littermates were fixed in 4% paraformaldehyde at 4°C for 24 h, washed in PBS, decalcified in 0.5 M EDTA (pH 8.0) for a week or a month (depending on mouse age), dehydrated in graded ethanol solutions, cleared in xylene and embedded in paraffin. Five-micrometer sections were cut, stained with Safranin O and immunohistochemically stained using standard protocols. Antibodies against collagen type X (1:50; BIOCYC, Luckenwalde, Germany), pERK1/2 (1:500; Cell Signaling Technology, USA) and Ki67 (also known as Mki67; 1:3000; Abcam, Cambridge, MA, USA) and the Dako Envision kit (Dako North America, CA, USA) were used. Images were acquired with a PD70-IX2-UCB microscope (Olympus, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Mean areas of individual HCs and number of HCs were measured from collagen X-labeled sections, within multiple 8500 µm² regions of interest on sections immunostained for collagen type X.

FA and ASP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stearic acid, Poloxamer 188, chitosan and lecithin was obtained from Spectrum Chemicals (Gardena, CA, USA). Dichloromethane (DCM) was obtained from Fisher Scientific (Houston, TX, USA). Sodium chloride was purchased from ChemCruz (Santa Cruz, CA, USA). Hydrochloric acid was purchased from Ricca Chemicals (Arlington, TX, USA). The primary antibodies against PCNA, p-ERK1/2 (Thr202/Tyr204), and p-RB were obtained from Cell Signaling Technologies (Danvers, MA, USA). The MKI67 and p21 primary antibodies were purchased from Abcam (Cambridge, MA, USA).

Human and mice skin tissues were fixed with 4% paraformaldehyde in PBS, embedded in paraffin, sectioned, and stained with H&E for histopathologic examination. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded skin tissues. The slices were probed overnight with SerpinB7 (Sigma-Aldrich, HPA024200), mKi67 (Abcam, ab16667). For Immunofluorescence, skin frozen sections were cryosectioned, blocked, and then stained with anti-Filaggrin (Biolegend, 905804), anti-KRT10 (Abcam, ab76318), anti-Loricrin (Biolegend, 905101). Images were captured using an Olympus BX600 microscope (Olympus Corporation, Tokyo, Japan) and SPOT Flex camera (Olympus Corporation, Tokyo, Japan) and were analyzed with ImagePro Plus (version 6.0, Media Cybernetics) software. The epithelial thickness and infiltrating cells were calculated in independent regions as described previously [47 (link)].

PMSCs at passage 5 were fixed in 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After 120 min blocking with 3% BSA (SIGMA), cells were incubated with primary antibody overnight at 4°C. The next day, cells were washed and stained with secondary antibodies (1:300, goat antirabbit IgG-Cy3; or 1:300, goat antimouse IgG-FITC) for 60 min at room temperature and then washed three times with phosphate-buffered saline (PBS). The primary antibodies for respective cells include PRDM1 (1:100, Abcam), CXCL8 (1:300, SANTA CRUZ), TOP2A (1:200, Abcam), MKI67 (1:100, Abcam), DEDD2 (1:100, Abcam), THY1 (1:100, Abcam), CITED2 (1:100, Abcam), and IGFBP6 (1:100, Abcam). Cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (1:500). The images were captured using Olympus IX73 and further analyzed with ImageJ software.

Cultured cells or tissue were fixed in 4% paraformaldehyde for 11 min followed by blocking in PBS with 2.5% normal goat serum, 0.3% triton X-100, and 2% bovine serum albumin for 30 min Primary antibodies used were Keratin 1 (Biolegend: PRB-149P) at 1:1000, Filaggrin (Abcam: ab3137) at 1:200, MKi67 (Abcam: ab16667) at 1:300, Keratin 10 (Abcam: ab9025) at 1:500, HNRNPK (Bethyl Laboratories: A300–674A) at 1:1000 for 1 h. The secondary antibodies used were Alexa 555 conjugated goat anti-mouse IgG (ThermoFisher: A11029) or Alexa 488 conjugated donkey anti-rabbit IgG (ThermoFisher: A21206) both at 1:500. Nuclear dye, Hoechst 33342 was used at 1:1000 (ThermoFisher: H3570).

Formalin‐fixed, paraffin‐embedded (FFPE) tissue sections were deparaffinized and rehydrated by using xylene and ethanol, respectively. After retrieval with citrate buffer at pH 6.0, the endogenous peroxidase activity of specimens was quenched by peroxidase‐blocking solution (Dako). The sections were incubated with the primary antibody against the target protein as indicated below: MYC (Abcam, ab32072), CEBPD (Abcam, ab184911), HK2 (Cell Signaling, 2867), FBXW7 (Abcam, ab105752), phospho‐AKT (Cell Signaling, 4060), phospho‐mTOR (Abcam, ab51044), phospho‐4E‐BP1 (Cell Signaling, 9644), phospho‐RPS6 (Abcam, ab80158), and MKI67 (Abcam, ab66155), followed by the incubation of a secondary antibody (REAL EnVision/HRP, rabbit/mouse [ENV], Dako). Staining was visualized with EAL DAB+ Chromogen diluted in REAL Substrate Buffer (Dako). Hematoxylin was used for the nuclear stain. Finally, the slices were dehydrated by soaking in various concentrations of ethanol and were mounted in UltraCruz Aqueous Mounting Medium with DAPI. The IHC staining results were examined with an optical microscope and quantified into H‐scores by three expert pathologists (Chien‐Feng Li, Tzu‐Ju Chen and Wan‐Shan Li ) as previously mentioned.²² Detailed information is shown in Supporting Information.