Diaminobenzidine substrate kit

Formalin-fixed, paraffin-embedded sections were cut into a thickness of 4 μm and mounted on Matsunami adhesive silane-coated glass slides (Matsunami Glass, Osaka, Japan). After deparaffinization and rehydration, the slides were autoclaved in 10 mM citrate buffer for 20 min to retrieve the antigens. Then, endogenous peroxidase was quenched with 0.3% hydrogen peroxide (H₂O₂) in methanol at room temperature for 10 min. After blocking, the sections were incubated at 4 °C overnight with the following primary diluted antibodies: anti-CYP2E1 (dilution, 1:500; Abcam, Cambridge, UK), anti-RIPK1 (dilution, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-RIPK3 (dilution, 1:300; Imgenex, San Diego, CA, USA). Subsequently, the sections were incubated with peroxidase-labeled polymer conjugated secondary antibody (Dako Japan, Tokyo, Japan) for 30 min at room temperature. Immunoreactivity was detected with a diaminobenzidine substrate kit (Dako Japan), and the sections were counterstained with hematoxylin. ImageJ imaging analysis software (National Institutes of Health, Bethesda, MD, USA) was used to quantitate the percentage of necrotic area. Field images at ×100 magnification were selected at random from different individuals. The percentage of necrosis was determined by measuring the total dimension of the field and comparing it with the dimension of the necrotic area.

All the mice were euthanized at 3 days after the last injection. For immunohistochemical analyses, all the tumor tissues were embedded in paraffin and dissected into 4-μm sections. To assess the proliferation of tumor cells, tissue sections were incubated with primary monoclonal antibody against Ki-67 (1:200; CST, USA) overnight after blocking with goat serum for 1 hour. Then, tissue sections were incubated with horseradish peroxidase–conjugated secondary antibodies using a diaminobenzidine substrate kit (Dako, Glostrup, Denmark). The histology of tumor tissues was examined under an optical microscope (DP73; Tokyo, Japan).

Samples from 24 patients (2 cases were lacking the epithelial component in the IHC test) with OKCs were fixed in 4% paraformaldehyde and embedded in paraffin under the guidelines of the National Institutes of Health. The tissue samples and CFMPs used for the corollary analyses were collected from the same patient. In brief, the sections were dewaxed, rehydrated and antigen-retrieved by high pressure. Subsequently, the sections were incubated with 3% hydrogen peroxide for 20 min, goat serum for 20 min under room temperature, followed by incubation with the primary antibody [receptor activator for nuclear factor-κB ligand (RANKL), 1:200, Proteintech Group, Wuhan, China; 23408-1-AP] at 4°C overnight. For the staining, a diaminobenzidine substrate kit, and hematoxylin (both from Dako, Glostrup, Denmark) were used. For RANKL evaluation, the semi-quantitative analysis of immunohistochemical staining was performed using Image-Pro Plus 6.0 and the quantification was calculated as the mean density for each protein (IOD/area).