Envision detection systems peroxidase dab

All paraffin-embedded tissues were sectioned at 4 μm, and the workflow was carried out as the manual of EnVision™ Detection Systems Peroxidase/DAB (Dako, Denmark, #K5007). Heat-induced technique was used for the epitope retrieval, followed by primary and secondary antibodies incubation. DAB developer and hematoxylin staining were applied in turn. PBS instead of the primary antibodies were negative controls [14 (link)]. Ventana ALK assay (Predilute D5F3 antibody) were employed to detect the ALK rearrangement on Ventana Benchmark XT platform using the in-house validated protocol [20 (link)].
Two pathologists evaluated the IHC staining blindly without any knowledge of the patients’ clinical information in a semiquantitative method. In each slide, ten areas were randomly selected under light microscopy, and scored for both of the quantity and intensity of positively stained cells. Quantity was scored as 0 for no staining; 1 for < 20% cells stained; 2 for 20–50% cells stained; and 3 for > 50% cells stained; whereas intensity was referred to as 0 for no appreciable staining; 1 for barely detectable staining; 2 for readily appreciable brown staining and 3 for dark brown staining. Quantity scores multiplied by intensities were the total scores, and 0–2 scores were negative, while positive for the others [14 (link)].

Immunohistochemistry (IHC) staining was carried out using EnVision Detection Systems Peroxidase/DAB (DAKO, Shanghai, China) following the manufacturer’s recommendations. Slides containing the sections were stained with commercially available anti-USP9X (ab180191, 1:500, Abcam), anti-EGLN3 (ab238941, 1:500, Abcam) and anti-Ki67 (ab15580, 1:500, Abcam). Two experienced pathologists scored the stained tissues independently. By recording the percentage of positive staining (0 = negative, 1 ≤ 10%, 2 = 10–50%, 3 ≥ 50%) and staining intensity (0 = no, 1 = weak, 2 = moderate, 3 = strong) for each sample, immunoreactivity score (IRS) (0–9) was calculated by multiplying positive staining percentage with staining intensity. Low and high expression were defined according to the median IRS.

Specific IgM and IgG for the SARS-CoV-2 membrane protein in COVID-19 patients sera were tested in Western blot. M protein was resolved on (4/14%) SDS-PAGE under reducing conditions (Mini Protean System, Bio-Rad, CA, USA). After semi-dry transfer to a nitrocellulose membrane (ThermoFisher Scientific, Waltham, MA, USA), the membrane was blocked in blocking buffer (1% BSA/10 mM PBS, 150 mM NaCl, 0.05% Tween-20, pH 7.2) for 1 h at room temperature. Randomly selected sera samples from COVID-19 convalescents and control subjects, previously tested in ELISA as described above (diluted 1:50 in blocking buffer) were individually incubated with membrane overnight on 4 °C. Membranes incubated in blocking buffer, omitting serum, were used for assessment of nonspecific binding. Incubation with sheep anti-human IgM/HRP or anti-human IgG/HRP conjugate (INEP, Belgrade, Serbia), diluted 1:1000 in blocking buffer, was carried out one hour at room temperature. Following each incubation step, membranes were washed in PBST. DAB staining was used (EnVision Detection Systems Peroxidase/DAB, DAKO, Santa Clara, CA, USA). Membranes were washed in water, dried, and scanned.