Gb111141

For immunohistochemical staining with positive cells of Ki67, alpha smooth muscle actin, smooth muscle myosin heavy chain, and desmin, paraffin-embedded sections of tissue underwent antigen retrieval by incubation in citrate buffer for 5 min at 85°C. Following 5% normal goat serum incubation, the sections were treated with antibodies specific to Ki67 (GB111141, Servicebio), alpha smooth muscle actin (GB111364, Servicebio), smooth muscle myosin heavy chain (GB11805, Servicebio), and desmin (GB11081, Servicebio) overnight at 4°C. After incubated with secondary antibodies for 1 h at room temperature, the section samples were stained with avidin-biotin complex and counterstained with hematoxylin. For TUNEL assay, the TdT-mediated dUTP nick end labeling kit (GDP1041, Servicebio) was performed in the aortic tissue to determine TUNEL-positive staining. The stainings were examined, and images were captured under a microscope (Nikon).

The tissue samples were collected and embedded in OCT (Q800, TA, USA). Five-micrometer-thick cross-sections were cut from the OCT-embedded samples, followed by fixation with cold acetone. Hematoxylin and eosin (HE) staining and Masson staining were performed for histological evaluations. The immunohistochemical staining of Ki67 (GB111141, Servicebio, 1:500), ARG1 (bs-23837r, Bioss, 1:500), iNOS (GB1119, Servicebio, 1:500) and CD31 (GB12063, Servicebio, 1:500) was performed to determine the proliferative cells, macrophage polarization and the formation of new vessels, respectively. The expression levels of iNOS and ARG1were further quantified by calculating the mean optical density via ImagePro Plus 6.0 software. Specifically, the integrated optical density (IOD) of the positive area (Area) of each image was measured and accumulated to obtain the IOD SUM. The mean optical density = (IOD SUM)/Area, which reflected the immunoreaction intensity in this image.

Immunohistochemical staining was performed as previously (15 (link)). Primary antibodies tested included those for SIRT1 (8469; Cell Signaling Technology), p65 (8242; Cell Signaling Technology), F4/80 (GB11027, Servicebio, Wuhan, China), and Ki67 (GB111141, Servicebio, Wuhan, China). For quantification, randomly selected 10 fields from each tissue section were evaluated to determine the percentage of positive staining cells per square millimeter.

Intestine and colon tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (HE) following standard protocol for histopathological analysis. Immunohistochemistry was performed to determine the protein expression of CEACAM5. Briefly, tissue sections were blocked with blocking buffer (Beyotime, P0260) for 30 minutes at room temperature. Then slides were incubated overnight at 4°C with the primary antibodies of CEACAM5 (Abclonal, A12421). After rinsing with PBS, slides were incubated for 30 min at 37°C with HRP Polymer Conjugate (ZSGB-BIO) and observed by microscopy.
Immunofluorescence was used to determine the degree of proliferation in CD4+ T cells. After blocked as previously described, slides were incubated overnight at 4°C with the primary antibodies of CD4 (Servicebio, GB13064-2) and Ki67 (Servicebio, GB111141). The slides were incubated with secondary antibodies (Alexa Fluor^®647-conjugated goat anti-rabbit IgG, bs-0296G-AF647; Dylight-550 Goat Anti-rabbit IgG secondary antibody, BA1135) for 1 h at room temperature followed by washing three times with PBST. After counterstaining nuclei with 4’,6-diamidino-2-phenylindole (DAPI), slides were imaged using a fluorescence microscopy (Nikon Eclipse C1). Staining results were evaluated by two independent observers.

Paraffin-embedded tissue sections (3 μm) were deparaffinized and rehydrated in a graded series of alcohol concentrations. The following primary antibodies were used for immunohistochemistry (IHC): α-SMA (1:50, rabbit polyclonal, Abcam, ab5694), GFP (1:100, rabbit polyclonal, Abcam, ab183734), and ki67 (1:600, rabbit polyclonal, Servicebio, GB111141). Sections were stained with a Sirius Red/Fast Green (Chondred) and antibody for IHC or H&E using routine protocols.
For immunofluorescence staining, liver tissue was fixed overnight at 4 °C in 4% paraformaldehyde, immersed in a graded series of sucrose solutions, embedded in OCT medium (Tissue Tek) and stored at -80 °C. Frozen sections were then prepared and incubated with the following antibodies: CD24 (1:200, mouse polyclonal, Biolegend, 101801; 1:200, mouse polyclonal, Santa Cruz, sc-19585), Alb (1:200, rabbit polyclonal, Proteintech, 16475-1-AP), CK19 (1:200, rabbit polyclonal, Proteintech, 10712-1-AP), Hnf4α (1:100, mouse polyclonal, Abcam, ab41898), and α-SMA (1:100, mouse polyclonal, Sigma, A5228). Representative images of Sirius Red, H&E, and IHC staining were captured with a Leica Aperio AT Turbo. Images of immunofluorescence staining were captured with a Leica TCS SP8. Sirius staining was quantified by Image J software.