Gk500710

Immunohistochemical staining was performed on the paraffin-embedded liver sections according to standard procedures. Liver sections were deparaffinized with 100% xylene, rehydrated with an ethanol gradient followed by 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol, and immersed in 3% H2O2 to remove endogenous peroxidase. After antigen retrieval, slides were incubated with the primary antibodies α-SMA (ab5694, Abcam, Cambridge, United Kingdom) and Col1a1 (BA0325, Boster, Wuhan, China) overnight at 4°C. Samples were then incubated with horseradish peroxidase-linked immunoglobulin G secondary antibody (GK500710, Gene Tech, Shanghai, China) at room temperature for 1 h. Staining was performed using an Envision Detection Rabbit/Mouse Kit (GK500710, Gene Tech).

Tissues were fixed with 4% (v/v) formaldehyde, embedded in paraffin and sectioned as described previously.37 Immunohistochemistry was performed with primary antibodies against Ki‐67 and CD34 (MAB‐0672 and Kit‐0004, MXB Biotechnologies) and an HRP–conjugated secondary antibody (GeneTech, GK500710). In negative controls, the primary antibody was replaced with 5% BSA. The stained sections were examined under a light microscope (Leica DM2000) with a digital camera (Olympus, DP73).

Immunohistochemistry was performed on tissue section microarrays (Zhuolibiotech, #ZL-Brc Sur122). The staining procedure included heat-induced epitope retrieval using 0.1 M sodium citrate (pH 9.0, heated by microwave to 90-95 °C, 3 times for 5 minutes each), incubation with primary antibody at 4 °C overnight. Signal detection was performed using an IHC detection kit (Gene Tech, #GK500710). Microscopy of the immunostaining included an initial pre-screen at low power (4X) to identify regions with a technically optimal staining result. Subsequently, detailed analysis at high-power (40X) was performed to evaluate the staining according to routine algorithms employed in tumor diagnostics. Scoring of FREM1 staining were evaluated using the semi-quantitative immunostaining score (ISS) method by pathologist. The immunostaining score was defined as 0 – 3 (range: +++/3, high; ++/2, moderate; +/1, weak; 0, negative.) [40 (link)]. The median score was used as cut off for classification of patients into high- and low- expression groups. Semi-quantitative analysis of the IHC images was conducted by Image-J, by which integral optical density (IOD) and the area were collected. Then, average optical density (AOD) was calculated as IOD/area, which represented the staining intensity [41 (link)].

Paraffin-embedded tissue specimens were sliced into 4-μm sections (LEICA, Germany) and heated at 60 °C for two hours. Then, the sliced specimens were dewaxed in xylene and rehydrated in graded ethanol solutions following standard procedures. Citrate buffer (10 mM) was used for antigen retrieval, and 3% H₂O₂ was used to block endogenous peroxidase activity. The slides were incubated with primary antibodies (anti-FLNC–1:250, anti-p-FLNC–1:400, anti-AKT1–1:500, anti-ERK1/2–1:250, anti-p-ERK1/2–1:200, anti-JNK–1:100, anti-p-JNK–1:50, anti-P38–1:100, and anti-p-P38–1:500) at 4 °C overnight. After incubation with the secondary antibody (GK500710, Gene Tech, China) at room temperature for 30 min, the slides were treated with DAB substrate. The staining images were acquired under an Olympus BX51 microscope.

Liver tissues were excised and fixed with 4% paraformaldehyde in PBS for 24 h and then dehydrated in PBS containing 30% sucrose for at least 16 h. Following paraffin embedding, the tissue sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed as described previously with modifications (Wu et al, 2011 (link)). Briefly, sections were deparaffinized with xylene and rehydrated. Heat‐induced epitope retrieval was performed by placing slides immersed in sodium citrate buffer (pH 6.0) at 100°C for 20 min followed by endogenous peroxidase blocking with 3% H₂O₂ for 30 min. Blocking was performed with 2% (w/v) BSA, and then the sections were incubated with Ki67 antibody (CST, 9449, 1:400) at 4°C overnight. Subsequently, the sections were incubated with Horseradish peroxidase (HRP)‐conjugated goat anti‐mouse antibody for 2 h at room temperature, and then stained by diaminobenzidine tetrahydrochloride (Gene Tech, GK500710). The sections were counterstained with hematoxylin.