Diaminobenzidine dab

After deparaffinization and rehydration, the femurs were immersed in citrate buffer (pH = 6.0). This was followed by intervention with 3% H₂O₂ and washing with PBS for 5 min three times. Then, the femurs were blocked with normal goat serum (3%) at 37 °C for 30 min and treated with the primary rabbit anti-rat antibody CD41 (1:100, Proteintech, Wuhan, China), VWF (1:100, Proteintech, Wuhan, China) overnight at 4 °C. Afterwards, they were treated with an enzyme-labeled goat anti-rabbit IgG secondary antibody for 60 min and stained with diaminobenzidine (DAB) (Servicebio, Wuhan, China); the sections were mounted, dehydrated, cleared, and counterstained with hematoxylin for 3 min. Finally, images were observed and photographed under an Olympus BX51 microscope (Olympus Optical).

Ki67 immunostaining was performed to evaluate the proliferative ability of the impaired tissue. The wound tissue was fixed with 4% paraformaldehyde fixation solution for 24 h at RT and cut into sections. Immunocytochemistry was performed on the paraffin sections, and the sections were blocked with immunostaining blocking solution (Biyuntian, Shanghai, China) at RT for 30 min and then incubated with an anti‐Ki67 antibody (1:200, Abcam, Cambridge, UK) at 4 °C overnight. Next, the sections were rinsed and incubated with an HRP‐conjugated anti‐rabbit IgG (1:600, Servicebio, Wuhan, China), followed by diaminobenzidine (DAB, Servicebio, Wuhan, China) and weak counterstaining with diluted hematoxylin (Servicebio, Wuhan, China) for 30 seconds. Images of immunohistochemical staining in the sections were acquired using a Leica microscope.

The samples were fixed in 4% paraformaldehyde solution for 24h, decalcified in 0.5 mol/L ethylene diamine tetraacetic acid (EDTA) for 6 weeks. After dehydrated and embedded in paraffin, the samples were cut into 4-μm sections for staining. For histological analysis, the sections were stained with hematoxylin and eosin (HE) as well as Masson’s trichrome stain according to the manufacturer’s protocol (Servicebio, China). For immunohistochemistry analysis, the sections were incubated with primary antibodies at 4°C overnight after dewaxed, rehydrated, and antigen retrieval. The primary antibodies were as follows: polyclonal rabbit anti-OCN antibody (1:200, Abcam, ab93876) and polyclonal rabbit anti-OPN antibody (1:200, Abcam, ab8448). After incubated with the corresponding secondary antibodies (Invitrogen, USA) for 30 min at room temperature, the signals were detected by diaminobenzidine (DAB) (Servicebio, China) and counterstained with hematoxylin (Servicebio, China). Finally, all tissue slices were photographed by the Aperio AT2 slide scanner (Leica Biosystems, Germany). The immunohistochemistry images were assessed by ImageJ with the IHC-Toolbox plugin (NIH, USA).

The immunohistochemistry (IHC) staining of tissues was performed as previously described [33 (link)]. The primary antibody used was the anti-FPR (1:500; Cell Signaling Technology, Danvers, MA, USA). The secondary antibody used was biotin-conjugated anti-rabbit IgG (1:200; Servicebio, Wuhan, China). The staining was visualized using diaminobenzidine (DAB; Servicebio, Wuhan, China), with brown cells indicating a positive result.
The immunofluorescence staining of the OFs was performed as previously described [34 (link)]. The primary antibodies used were the FPR (1:500; Cell Signaling Technology, Danvers, MA, USA), vimentin (1:500; Abclonal, Wuhan, China), cytokeratin (1:500; Abclonal, Wuhan, China), desmin (1:500; Abclonal, Wuhan, China), S-100 (1:500; Abclonal, Wuhan, China), and myosin (1:500; Abclonal, Wuhan, China). The secondary antibody was FITC-conjugated goat anti-rabbit IgG (H + L) (1:200; Servicebio, Wuhan, China).

Paraffin-embedded xenograft sections were used for TDP-43 (Servicebio, China, GB112160, dilution: 1:400), ABHD2 (absin, abs140798, dilution: 1:200), C-caspase3 (Servicebio, GB11532, dilution: 1:500), Bcl-2 (Servicebio, GB113375, dilution: 1:500), and p53 (Servicebio, GB111740, dilution: 1:500) staining analysis. The antibody of Ki67 used in this experiment was purchased from Abcam (ab15580, dilution: 1:200). After deparaffinized and rehydrated, sections were laid in EDTA solution (pH9.0) for antigen retrieval by boiling in microwave oven for 8 min on medium heat. The slides were naturally cooled before treating with 3% H₂O₂ for 10 min. Sections were blocked in 10% normal goat serum for 30 min at room temperature, and then incubated overnight with indicated primary antibody at 4°C. After three washes with PBS, the slides were hybridized with the secondary antibody (Servicebio, GB23303, dilution: 1:200) for 50 min at room temperature. Finally, diaminobenzidine (DAB) (servicebio) was used for color development. After counterstaining with hematoxylin, dehydrate the slides and seal with neutral gum (Solarbio, China). Image J software was used for staining analysis.

Tissue was also fixed in 10% formaldehyde. According to the standard protocol, the paraffin sections were deparaffinized. After repairing antigen and blocking endogenous peroxidase act, tissue sections were blocked in 3% BSA. Tissue sections were incubated with primary antibodies (Ki67, cleaved caspase3, cleaved PARP1, Servicebio, Wuhan, China) at 4 °C overnight, followed by conjugated secondary antibodies (Servicebio, Wuhan, China) and diaminobenzidine (DAB, Servicebio, Wuhan, China). Then nuclei counterstaining was performed with Mayers hematoxylin (Servicebio, Wuhan, China). Finally, the tissue sections were dehydrated and sealed.

To localize miR-126 expression in the tissue sections, a miRNAScope™ HD (RED) Assay Kit (Bio-techne, USA) was used according to the manufacturer’s instructions and established protocols. Briefly, after dewaxing, hydrogen peroxide blocking, and heat-mediated antigen retrieval using a steamer, Protease III was applied. After the hybridization probe and Hybridize Amp1-6 were added dropwise in sequence, diaminobenzidine (DAB, Servicebio, China) was used for visualization of miR-126, and the slides were then counterstained and mounted. Scrambled miRNA was used as a negative control.