Envisiontm system

The dissected colon tissues were prepared for immunohistochemical (IHC) analysis of the expression patterns of Nrf2, HO-1, GCLM, and GPx-2. Four-lm sections of 10% formalin-fixed, paraffin-embedded tissues were cut on silanized glass slides and deparaffinized three times with xylene and rehydrated through graded alcohol bath. The deparaffinized sections were heated by using microwave and boiled twice for 6 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. To diminish nonspecific staining, each section was treated with 3% hydrogen peroxide and 4% peptone casein blocking solution for 15 min. For the detection of respective protein expression, slides were incubated with Nrf2 (Bioworlde, USA), HO-1 (Cell Signaling Technology, USA), GCLM (Santa Cruz Biotechnology), and GPx-2 (Santa Cruz Biotechnology) antibodies at room temperature for 40 min in Tris-buffered saline containing 0.05% Tween 20, and then developed using respective horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit, mouse, or goat) EnVisionTM System (Dako). The peroxidasebinding sites were detected by staining with 3,3′-diaminobenzidine tetrahydrochloride (Dako). Finally, counterstaining was performed using Mayer’s hematoxylin.

After sacrifice, hindlimbs/tumors were collected to perform a histopathologic characterization. Paraffin sections (3 μm thick) were cut, dewaxed and hydrated. Sections of all tumors were stained with hematoxylin and eosin for histologic evaluation. Immunohistochemical staining for Ki-67 (rabbit monoclonal, clone SP6, 1:100, NeoMarkers; RM-9106) was performed on those tumors/hindlimbs of treated mice, using the EnVisionTM+ System (Dako, Glostrup, Denmark) according to the manufacturer’s recommendations.

Tumor sections of bladder cancer patients or nude mice xenografts were studied by immunohistochemistry (IHC) assay using EnVisionTM System (DAKO, Carpinteria, CA, USA). Primary antibodies used in IHC were KLF5 (Abcam, ab24331, 1:200), PCNA (Santa Cruz, sc-7907, 1:300), VEGFA (Abcam, ab46154, 1:150) and CD31 (Epitomics, 2530-1, 1:100). Staining signals were photographed using an Olympus BX51 Microscope (Olympus, Tokyo, Japan). Average intensity score of the positive cells (0-none, 1-weak, 2-intermediate, and 3-strong) and percentage score of stained cells (1–0% to 25%, 2–25% to 50%, 3–50% to 75%, and 4–75% to 100%) were multiplied to get the total staining score, ranging from 0 to 12. Neovessel number in a bladder cancer tissue was counted following the previously described method [36 (link)].

NSCLC tissue specimens from 2015 to 2017 were used. These patients underwent tumour resection at Taihe Hospital. The tumour tissues were fixed in 4% formalin and embedded into tissue blocks. Serum samples were also collected from NSCLC advanced patients and healthy donors. The serum was stored in a −80 °C freezer. Immunohistochemistry analyses were performed using a DAKO EnVisionTM system (Dako, Glostrup, Denmark). Experimental steps were carried out according to the appropriate instructions. The following antibodies were used: KEAP1 from Abcam (Cambridge, MA, USA). Semi-quantitative analysis was used for the tissue staining results. The intensity was scored as follows: 0 negative; 1 weak; 2 moderate; and 3 strong. According to the degree of staining, negative and weak were considered low expression, and moderate and strong were considered high expression.

Paraffin-embedded JAK2wt and JAK2V617F⁺ EBs were prepared as described [80 (link)]. Nonspecific binding sites were blocked with 0.2% bovine serum albumin for 1 h, sections stained with primary antibody at RT for 1 h. EnVisionTM system (Dako, Glostrup, Denmark) including horseradish peroxidase and diaminobenzidine (DAB⁺, Dako) was used for antibody detection; cell nuclei were counterstained by hematoxylin. Images were acquired using an Olympus BX51 inverted microscope equipped with ColorViewIII digital CCD camera.