Horseradish peroxidase conjugated anti rabbit antibody

Nigericin and LPS were purchased from InvivoGen Biotech Co., Ltd. (San Diego, CA, USA). Mouse anti-FLAG, mouse anti-myc, mouse anti-β-actin, mouse anti-HA, rabbit anti-IL-1β, rabbit anti-NLRP3, rabbit anti-Casp-1, and rabbit anti-ASC monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rabbit antibody or anti-mouse antibody were purchased from ZSGB-BIO, Lnc. The translation inhibitor cycloheximide (CHX) was purchased from APEXBIO (Houston, TX, USA). Lipofectamine 2000 was purchased from invitrogen (Waltham, MA, USA).

Following formalin fixing, paraffin‐embedded xenografted tumor tissue sections were deparaffinized in xylene and rehydrated with decreasing grades of ethanol. The slides were preincubated with 3% hydrogen peroxide methanolic solution for 30 min at room temperature and heat‐induced epitope retrieval was carried out. After blocking with goat serum for 2 h, slides were incubated with rabbit anti‐DDR1 (Novus Biologicals) 1 : 100 diluted at 4 °C overnight. Following a wash, slides were incubated with horseradish peroxidase‐conjugated anti‐rabbit antibody (Zsbio) for 30 min at 37 °C and then visualized using diaminobenzidine tetrahydrochloride.

Paraffin tissue sections were deparaffinized in xylene and rehydrated gradually in serial dilutions of ethanol. Antigen retrieval was performed by autoclave treatment at 95°C for 3 min in 0.01M citrate buffer (pH = 6.0, ZSGB-BIO, Beijing). The slides were then incubated with 0.3% H₂O₂ for 10 min to quench endogenous peroxidase activity. After washed with PBS three times, the sections were blocked with goat serum for 15 min, followed by incubation with polyclonal rabbit anti-ROR1 antibody (1:20, Abcam, England) at 4°C overnight. After wash, sections were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (ZSGB-BIO, Beijing) at 37°C for 15 min. Negative controls were obtained by replacing the primary antibody with PBS. The sections were stained with DAB⁺ substrate-chromogen solution (Maixin Biotech. Co., China). Counterstaining was performed with hematoxylin. ROR1 immunostaining was independently scored by two experienced individuals according to intensity and percentage of ROR1-positive cells. Staining intensity was scored as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The percentage of ROR1 positive cells was divided into 4 categories: 1 was for 0–10%, 2 for 11–50%, 3 for 51–80%, 4 for 81–100%.