Vectastain elite abc universal plus kit

Human spinal cord paraffin sections from clinically and histopathologically characterized ALS patients and neurologically healthy individuals were obtained from the Sheffield Brain Tissue Bank. Consent was obtained from all subjects for autopsy, histopathological assessment, and research in accordance with local and national Ethics Committee–approved donation. Three control cases, three sALS, and three ALS-C9 cases were included in the study. Human spinal cord sections were 7 µm thick. Rehydrated sections were subjected to microwave antigen retrieval in sodium citrate buffer. After incubation with the primary anti-UBAP2L antibody overnight at 4°C, secondary HRP-conjugated anti-rabbit IgG antibody (Vector Laboratories) was applied for 1.5 h at RT. Vectastain Elite ABC Universal Plus Kit (Vector Laboratories) and DAB (Sigma-Aldrich) were used for detection. Images were taken with a 20× objective (UPLFLN 20×Ph/0.5) of a BX53 microscope equipped with DP73 camera and processed using cellSens Standard 1.9 software (all Olympus).

Tissue microarray slides containing 140 prostate cancer and benign prostate tissue sections were gifts from Dr. Jiaoti Huang (Duke University, Durham, NC, USA). After deparaffinization and hydration, tissue slides were treated with 3% H₂O₂ for 15 min and then blocked with 5% bovine serum albumin (BSA) in Tris-buffered solution with Tween-20 (TBS-T) for 60 min at room temperature. The primary antibody for MAPK8IP2 was purchased from Sigma-Aldrich (catalog #HPA034780) and used at 1:40 dilution in 5% BSA/TBS-T overnight at 4°C with agitation. Visualization of the immunosignals was performed using the VECTASTAIN® Elite® ABC Universal PLUS kit (Vector Laboratories, Inc., Burlingame, CA, USA). The immunosignal index was calculated by multiplying the immune density (weak = 1, moderate = 2, and strong = 3) with the percentage positivity, as described in our recent publication.36 (link)

From each tissue microarray block, 4‐μm sections were deparaffinized and rehydrated. The sections were then incubated with citric acid (pH 6.0) for 20 minutes at 100°C. The sections were stained with an anti‐VEGFR2 antibody (polyclonal, ab2349, 1:200; Abcam, RRID: AB_302998) at 4°C overnight. Mouse skin tissue was used as the positive control. The negative controls were treated with Tris‐buffered saline instead of primary antibodies. Following incubation with the secondary antibodies for 30 min at room temperature (approximately 25°C), the specimens were stained using the VECTASTAIN Elite ABC Universal PLUS Kit (Vector Laboratories, Inc.). All sections were counterstained with 100% hematoxylin for 30 seconds at room temperature (approximately 25°C). All immunohistochemistry results were assessed by an experienced pathologist. Immune cells staining (IC‐VEGFR2), including lymphocytes, macrophages, granulocytes, dendritic cells, plasma cells, and tumor cells staining (TC‐VEGFR2), was separately evaluated. IC‐VEGFR2 was defined as positive when any immune cells were stained with an anti‐VEGFR2 antibody in the tumor stroma or intratumor area. TC‐VEGFR2 was defined as positive when any tumor cells were stained with an anti‐VEGFR2 antibody in the specimen (Figure 1).