The paraffin-embedded sections were stepwise dewaxed from xylene, gradient ethanol to water, and performed immunohistochemical staining as protocol. IHC staining was performed with heat-induced antigen retrieval, followed by incubating in primary antibodies overnight at 4 ℃, secondary antibody (EnVision/HRP kit, DAKO) 30 min at RT (room temperature) and substrate-chromogen solution (DAB detection kit, DAKO) 5–10 min at RT, with hematoxylin counterstain for 10 s. Among each step, washing the slides in PBS three times for 5 min. Normal kidneys were used as positive controls; normal serum in place of the primary antibody as an isotype negative control, and the slides without added primary antibody were used as negative controls. The results were scored as previously described and scored as a sum of the staining intensity and percentage of positive tumor cells. Briefly, the staining intensity scaled with 0–3. Percentage of positive cells staining was scored as 0–4. The final staining was summed by intensity and percentage as 0–12. And then adapted to 4-point IRS: 0–1 (−), 2–3 (+), 4–8 (+ +), and 9–12 as (+ + +). Finally, we set “+“ as WTX expression cut off point: − as negative;+ ~ + + + as positive4 (link). The clinical pathologic meanings of the IHC data were analyzed by Chi-square analysis. IHC staining and statistics were performed in a blind manner.
Dab detection kit
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The DAB detection kit is a laboratory reagent used for the visualization of target proteins in immunohistochemistry and Western blotting applications. The kit contains the necessary components to perform a 3,3'-Diaminobenzidine (DAB) chromogenic reaction, which results in the deposition of a brown-colored precipitate at the location of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Envision hrp kit, by Agilent Technologies (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
Vectastatin abc kit, by Vector Laboratories (2 mentions)
Goat anti mouse igg hrp, by Agilent Technologies (2 mentions)
Goat anti rabbit igg hrp, by Agilent Technologies (2 mentions)
Optical microscope, by Olympus (2 mentions)
Tnf α, by Abcam (1 mentions)
Cd31 antibody, by BD (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Anti e cadherin, by Thermo Fisher Scientific (1 mentions)
Citrate buffer, by Agilent Technologies (1 mentions)
Ab18672, by Abcam (1 mentions)
Ab53519, by Abcam (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Second antibody, by Agilent Technologies (1 mentions)
Alexa fluor 488 phalloidin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Faramount aqueous mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
26 protocols using dab detection kit
1
Immunohistochemical Evaluation of WTX Expression
2
Immunohistochemical Analysis of Mouse Skin
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We performed H&E staining and immunohistochemical analyses using the paraffin sections of mouse skin biopsies with mAb against mouse granzyme B (clone TA312131, OriGene), IFNγ (clone bs-0480R, Bioss), or TNFα (clone ab6671, Abcam). The secondary antibodies conjugated to peroxidase and the DAB Detection Kit (Dako) were used for the following staining. The control slides were incubated with the secondary antibody, or isotype control antibodies alone.
3
Hepatic Cell Proliferation and Angiogenesis
4
Immunohistochemical Analysis of Mouse Tissues
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Primary tumors and lungs from mice were fixed with 4% paraformaldehyde, paraffin-embedded, and cut into 4-μm section slices. The slides were incubated in a dry oven at 60 °C for 1 h, de-paraffinized, re-hydrated, and incubated in a citrate buffer (Dako) at 121 °C for 10 min to retrieve antigen. To block non-specific signals. the slides were incubated with 3% H2O2 for 15 min and with 2% horse serum in BSA solution for 1 h. The slides were incubated overnight at 4 °C with anti-KHK (1:100, Santa Cruz), anti-LRRC59 (1:100, Novus Biologicals), or anti-E cadherin (1:200, Thermo Fisher Scientific). The sections were biotinylated with a secondary antibody for 1 h. The immune complexes were visualized using the Vectastatin ABC kit (Vector Laboratories) and the DAB detection kit (Dako). Finally, the slides were counterstained with hematoxylin for 15 min, and photographed at four high-power fields in each slide.
5
IHC Staining of IL-8, Snail, and Vimentin in HNSCC
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Immunohistochemical (IHC) staining was performed to detect IL-8, snail, and vimentin expression in HNSCC patient tissue samples. After deparaffinization and rehydration, the tissue slides were heated in a water bath at 100 °C with citrate buffer solution (pH 6.0) for 20 min to retrieve antigen, and then cooled at room temperature. The primary antibodies were incubated overnight at 4 °C in a humidified chamber and then visualized using a 3,3′-diaminobenzidine (DAB) detection kit (Dako Cytomation, Denmark) containing goat secondary antibody molecules and DAB chromogen. Every step of the wash used phosphate-buffered saline solution (PBS) for 5 min repeated three times. The primary antibodies (with their dilutions reported), including IL-8 (1:1000; ab18672, Abcam, USA), snail (1:1000; ab53519, Abcam, USA), and vimentin (1:200; D21H3, Cell Signaling Technology, USA) were used as the manufacturer’s instructions suggest. The intensity of the IL-8, snail, and vimentin immunoreaction was scored as follows: 0 = negative, absence of stained cells; 1 = weak; 2 = moderate; and 3 = strong. The IHC staining score was calculated by multiplying the percentage of positive cells by the staining intensity. The scoring was conducted by researchers who were blind to the clinical information of patients.
6
Histological Evaluation of Bladder Tissue
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5 μm sections were made after bladders being fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed to observe general morphology of the bladders, and Masson trichrome staining was used to evaluate the level of tissue fibrosis. Immunohistochemical staining was also conducted as previously described in our study. Briefly, the sections were subjected with 10 mM sodium citrate buffer (pH 6.0) to heat for antigen retrieval. The primary antibodies of anti-Nrf2 (Abcam, Cambridge, UK), HO-1 (Abcam, Cambridge, UK), PCNA (CST, Danvers, MA, USA), secondary antibodies of goat-anti-rabbit IgG-HRP (DAKO, Denmark), goat-anti-mouse IgG-HRP (DAKO, Denmark), and DAB detection kit (DAKO, Denmark) were used according to the manufacturer's instructions. Histological analysis was performed by a pathologist in a blinded manner.
7
Immunohistochemical Analysis of Protein Expression
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Immunohistochemistry staining was conducted as reported previously [21 (link)]. Briefly, the section was dewaxed, and the antigen was restored in EDTA buffer. Then, the section was incubated with 3% H2O2 in order to inhibit endogenous peroxidase. After blocking with 5% goat serum, the corresponding primary antibody was added onto the section at 4°C overnight, and then the section was incubated with the second antibody (Dako, Danish). After being treated with the DAB Detection Kit (Dako, Danish) and hematoxylin, two pathologists who were blind to the clinical information were invited to score the samples according to their staining intensity and the percentage of positive cells from 1 to 5. The score from 1 to 3 was regarded as a low expression group and the score from 4 to 5 was regarded as a high expression group. The primary antibodies used in this experiment are as follows: ZDHHC19 (1:200, CST, USA); Ki-67 (1:200, CST, USA).
8
Immunohistochemistry and Immunofluorescence Assays
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For immunohistochemistry, tissue slides were deparaffinized, rehydrated, and heated to retrieve antigen. The slides were sequentially incubated with 3% H2O2, 2% horse serum, primary antibodies, and biotinylated secondary antibodies. The slides were treated with the DAB detection kit (Dako), counterstained with hematoxylin, and photographed at four high-power fields for each slide. Protein expression was analyzed using histoscore (the staining intensity (0–3) × the percentage of positive cells). Human gastric cancer tissue arrays were obtained from SuperBioChips (Seoul, Korea). Clinical information on the patients is summarized in Supplementary Table 4 . For immunofluorescence, cells on a cover slide were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 and 0.05% Tween-20, and sequentially incubated with primary antibodies and Alexa Fluor 488-conjugated secondary antibodies (Invitrogen). F-actin was stained with Alexa Fluor 488 phalloidin (Abcam). After incubation with DAPI (Sigma‒Aldrich), the slides were mounted in Faramount aqueous mounting medium (Dako). Fluorescence images were observed under a confocal microscope.
9
Quantitative Immunohistochemical Analysis
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All tissue sections were stained according to the manufacturer’s instructions. Heat induced epitope retrieval was performed and a standard DAB detection kit was used for visualization of the immunoreaction (DAKO, Denmark). MMP9 and MMP2 expression were detected using a rabbit anti-mouse MMP9 and MMP2 polyclonal antibody (sc-393859 and sc-53630; Santa Cruz Biotechnology) diluted in 1:100. MPO and F4/80 expression were detected using a rabbit anti-mouse MPO and F4/80 polyclonal antibody (GB13027, GB11224; Servicebio, Wuhan, China), diluted in 1:800 and 1:2000, respectively. Sections were incubated at 4°C overnight with the primary antibodies and with biotinylated goat anti-rabbit IgG (KPL, Burlingame, CA) as secondary antibodies for 50 min at room temperature. Negative controls subjected to the same procedure without the primary antibody showed consistently negative results. MPO or F4/80-positive cells were counted by randomly selecting five high-power fields distributed over at least three independent sections.
The relative expressions of MMP9 and MMP2 proteins were quantified using the image-pro-plus 6.0 software (Media Cybernetics Company, USA). Five fields in each section were randomly chosen. The fraction of total intensity of brown substances (positive staining)/total area in each image (field) indicates the relative expression of the corresponding protein.
The relative expressions of MMP9 and MMP2 proteins were quantified using the image-pro-plus 6.0 software (Media Cybernetics Company, USA). Five fields in each section were randomly chosen. The fraction of total intensity of brown substances (positive staining)/total area in each image (field) indicates the relative expression of the corresponding protein.
10
Quantitative Immunohistochemical Analysis of AR and ER-α
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After the same process described above, the sections were incubated with rabbit polyclonal anti-AR antibody (1:80, Abcam) and rabbit polyclonal anti-estrogen receptor-α (ER-α) (1:100, Abcam) at 4°C overnight. After being washed in PBS three times, the sections were subsequently incubated with a secondary biotinylated anti-rabbit antibody (1:200, Aspen, Wuhan, China). The immunohistochemical staining was visualized with a DAB detection kit (DAKO, Glostrup, Denmark) and the sections were counterstained with Hematoxylin, dehydrated, cleared, and mounted. Sections incubated without primary antibody were also included in each staining experiment as a negative control to detect nonspecific binding of the secondary antibodies. Sections were assessed using an optical microscope (Olympus). IPP 6.0 software was used to measure the integral optical density (IOD) for quantitative analysis.
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