Envision plus system hrp

Immunohistochemistry studies of IER5 were performed on glioma samples in a TMA. The human glioma TMA (Product number: HBraG180Su01) was purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). Ethical approval was granted by ethics committee of Shanghai Outdo Biotech Company. The 180 cases of glioma in this microarray were from Chinese National Human Genetic Resources Sharing Service Platform⁴. And the platform number is 2005DKA21300. In brief, all the samples were incubated with an anti-IER5 antibody (Invitrogen, PA5-56287, United States; 1:300 dilution) overnight at 4°C. Afterward, the samples were incubated with HRP-labeled secondary antibodies for 1 h at 37°C. Finally, the samples were stained and imaged. EnVision Plus System-HRP (K8002, DAKO, Denmark) was employed, and IER5 was visualized with diaminobenzidine (DAB) as the substrate. The nucleus was stained with Mayer’s hematoxylin counterstain (GT100540, Gene). The assessment of IHC data was carried out by two readers and verified by two independent pathologists blinded to the clinicopathologic information. The staining intensity was defined as: 3 (strong), 2 (moderate), 1 (weak), and 0 (negative). Negative and weak staining intensity was classified as low IER5 expression, whereas strong and moderate staining intensity was classified as high IER5 expression.

Tissue sections from CRC specimens were deparaffinized in xylene, rehydrated through graded ethanol, and subjected to antigen retrieval by heating in sodium citrate (pH 6.0) in a microwave oven for 15 min. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide. After blocking with 20% goat serum for 30 min at room temperature, the sections were incubated overnight at 4°C with the primary antibodies (Table S5). The EnVision Plus System‐HRP (DAB; DAKO) was used according to the manufacturer's instructions, and the sections were counterstained with Mayer's hematoxylin.⁶⁵ The stained tumor sections were evaluated by two pathologists, scoring the percentage of positively stained tumor cells and staining intensity as described previously.⁶⁶

Bovine ovary sections (n = 4 from 8 cows) from a slaughterhouse were fixed, then deparaffinised, hydrated, and microwaved for 5 min in an antigen unmasking solution (Vector Laboratories Inc., AbCys), then allowed to reach room temperature. Sections (7 μm) were washed for 5 min in PBS then incubated in a peroxidase-blocking reagent for 10 min at room temperature in order to inhibit endogenous peroxidase activity (DakoCytomation). After two 5 min washes in PBS, the non-specific background was eliminated by blocking with 5% lamb serum in PBS for 20 min, followed by an overnight incubation at 4°C with PBS containing a rabbit primary antibody raised against CCL21 (1:100; Sigma, Aldrich, Saint Quentin Fallavier, France). Sections were washed in PBS twice for 5 min each, then incubated for 30 min at room temperature with a ready-to-use labeled polymer-HRP anti-rabbit antibody (EnVision Plus HRP system; DakoCytomation). The sections were washed in PBS twice, and staining was revealed after incubation at room temperature with 3,3-diaminobenzidine tetrahydrochloride (DAB) (Liquid DAB+ Substrate Chromogen System; DakoCytomation). The negative controls were prepared by replacing the primary antibodies with rabbit IgG.

Formalin-fixed, paraffin embedded (FFPE) ovarian cancer patient tissue blocks were obtained from the Department of Pathology, Erasmus Medical Center, Rotterdam. The average fixation time was 1–2 y. 4 μm sections were mounted on slides, dewaxed with Xylene (#28979.294, VWR Chemicals) and hydrated. Antigen retrieval was performed in Tris-EDTA buffer (pH 9.0) using a pressure cooker procedure. Slides were incubated in 3% hydrogen peroxidase (#95321, Sigma Aldrich) at RT for 10 min and blocked with 5% milk (#115363, Millipore) in PBS-Tween (#P1379, Sigma Aldrich) for 30 min. Immunohistochemistry was performed using antibodies directed against Tpm1.6/7 (1:100), Tpm1.8/9 (1:100) and mitochondria (1:100, #MAB1273, Sigma Aldrich) followed by the EnVision Plus-HRP system (Dako). Slides were incubated with primary antibodies O/N at 4 °C, washed twice with PBS-Tween and incubated with Rat EnVision+ System-HRP (#P0405, Dako) or Mouse EnVision+ System-HRP (#K4007, Dako) for 30 min. Slides were counterstained with Hematoxylin (#MHS16, Sigma Aldrich).

STAT3 and p-STAT3 (S727) were detected in the paraffin-embedded CCA tissues using a standard immunohistochemistry protocol. Briefly, the tissues were incubated with 1: 100 of anti-STAT3 or anti-p-STAT3 (S727) at room temperature, overnight. The Envision-Plus HRP system (Dako, Carpinteria, CA) against the primary antibody was applied at room temperature for 2 h. The peroxidase activity was observed using diaminobenzidine tetrahydroxychloride solution (DAB; Dako) as a substrate, and counter stained with hematoxylin. The frequency of target molecules were semi-quantitatively scored on the basis of percentage of the positively stained cells as 0 = negative; 1–25% = 1; 26–50% = 2; and >50% = 3. The intensity of stained cells was scored as weak = 1, moderate = 2 and strong = 3. The immunohistochemistry (IHC) index was calculated as frequency × intensity.