Mach1 universal hrp detection kit

Antigen retrieval was performed on 4 μm sections with heat‐induced epitope retrieval in a decloaking chamber (Nexgen, Biocare Medical, Concord, CA, USA) with sodium citrate (0.01 m, pH 6.0), 110 °C for 20 min. The MACH1 Universal HRP Detection Kit (Biocare Medical, LLC.) was used for visualisation. The non‐specific background staining was blocked using the MACH1 sniper blocking reagent (BioCare Medical) for 30 min, followed by overnight incubation with the NDRG1 antibody (1:250; Abcam, EPR5593). The slides were treated with MACH1 secondary antibody conjugated with universal horseradish peroxidase (HRP polymer) for 30 min at room temperature and were counterstained with haematoxylin.
The scoring for both patient cohorts was performed with a pathologist (AS). A positive stain was defined as >1% of cells displaying unequivocal staining, whereas a negative finding was defined as <1% positivity. The intensity of stained cells was scored as weak (1+), moderate (2+) and strong (3+). Additionally, the subcellular localisation pattern of protein expression was documented as either cytoplasmic or cytoplasmic + membrane (grouped under non‐nuclear staining); or nuclear and cytoplasmic ± membrane (grouped under nuclear staining).

Antigen retrieval was performed on 4 µm sections with heat-induced epitope retrieval in a decloaking chamber (Nexgen, Biocare Medical, Concord, CA, USA) with sodium citrate buffer (0.01 M, pH 6.0) at 110 °C for 20 min. The reagents from the MACH1 Universal HRP detection Kit (Biocare Medical, LLC, Concord, CA, USA, #M1U539 L10) was used for immunohistochemical detection. MACH1 sniper blocking reagent was used to block non-specific staining for 30 min. Primary antibodies against B7-H3 (1:200; Cell Signaling, #D9M2L/#14058, Beverly, MA, USA) and Ki-76 (1:100; Dako, #M7240, Santa Clara, CA, USA) were diluted in Biocare Da Vinci Green Diluent (Biocare Medical, #PD900, Concord, CA, USA) and incubated overnight at room temperature. The slides were treated with MACH1 secondary antibody conjugated with MACH1 Horse-Radish Peroxidase polymer for 30 min at room temperature. MACH1 diaminobenzidine substrate was applied for 5 min and the slides were counterstained with hematoxylin.