Streptavidin peroxidase ihc assay kit

GALNT6 expression in each tissue was examined by IHC. Briefly, tissue sections (3 µm) were deparaffinized, rehydrated, incubated with 3% H₂O₂ in methanol and subjected to antigen retrieval by EDTA buffer. The sections were blocked with 5% bovine serum albumin (BSA), probed with anti-GALNT6 (1:100, ab151329, Abcam), anti-GRP78 (1:100, ab21685, Abcam), or anti-phospho-ERK1/2 (1:100, #4370, CST) at 4 °C overnight. The sections were reacted with biotinylated secondary antibodies, and detected using the Streptavidin-Peroxidase IHC assay kit and DAB (ZSGB-bio, China). Immunostaining was evaluated by two certified pathologists in a blinded manner and scored, according to the staining intensity (negative staining: 0 point; weak staining: 1 point; moderate staining: 2 point; and strong staining: 3 point) multiplied by the percentage of stained cells (positive cells ≤ 25% of the cells: 1 point; 26–50% of the cells: 2 point; 51–75% of the cells: 3 point; ≥75% of the cells: 4 point). The median value of GALNT6 scores was employed to determine the cutoff. Tumors with GALNT6 scores lower or equal to the median were designated as“low expression”, whereas those with scores higher than the median were designated as “high expression”. The positive control of GALNT6 expression was defined according to lung cancer from the website (http://www.proteinatlas.org).

Human MSG tissues from 10 pSS patients and 7 Sicca control patients were obtained after informed consent. The gland tissues were fixed with 4% paraformaldehyde, followed with embedding in paraffin blocks. The slides were heated at 65°C for 30 min, followed by paraffin removal with xylene and subsequent rehydration with ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) at a sub-boiling temperature for 20 min. Samples were blocked with 10% goat serum for 1 h at RT and incubated with primary antibody (1:100) overnight at 4°C. The slides were visualized using streptavidin peroxidase IHC assay kit (ZSGB-bio, China) and counter stained with hematoxylin. The primary antibody of immunohistochemistry was performed using anti-IL-17A (Clone G-4, Santa Cruz), anti-CD4 (Clone EPR6855, Abcam), anti-p-STAT3 (Clone EP2147Y, Abcam)m and anti-p-STAT5 (Clone E208, Abcam). Images were obtained using an OLYMPUS-BX51 microscope at 10 × 10 or 40 × 10 magnification.

The resected specimens were obtained from primary lesions, fixed with formalin, embedded with paraffins. Serial 4 μm sections were prepared, then briefly incubated with xylene, rehydrated with graded ethanol solutions, incubated with methyl alcohol containing 3% hydrogen peroxide and immersed in a citrate buffer for antigen retrieval. IHC was performed using Streptavidin-Peroxidase IHC assay kit (ZSGB-bio, China) following the manufacturer’s instructions. Antibodies of Aurora A and YAP diluted 1:200 in PBS containing 2% goat bovine serum respectively. Immunostaining was evaluated by two pulmonary pathologists using a blind protocol design. For each specimen, the total score of intensity expression (negative staining: 0 point; weak staining: 1 point; moderate staining: 2 point; and strong staining: 3 point) and multiplying stained cell numbers (positive cells as ≤25% of the cells: 1 point; 26–50% of the cells: 2 points; 51–75% of the cells: 3 points; >75% of the cells: 4 points) of Aurora A or YAP was estimated. When the sample was scored ≥ 6 points, we defined it as high expression, otherwise low expression.