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The tissue sections were deparaffinized, rehydrated, blocked with 10% normal goat serum, and incubated with the anti-TMEM147 (Abcam, ab97624; 1:200 dilution), anti-4HNE (Abcam, ab46544; 1:250 dilution), and anti-ARG1 (Abcam, abab96183; 1:250 dilution) primary antibodies overnight at 4 °C. The slides were then incubated sequentially, first with a secondary antibody (Vector lab, Burlingame, CA, USA) for 1 h and then with Vectastain Elite ABC reagent (Vector Lab) for 30 min. The tissue sections were stained with diaminobenzidine (DAB kit; Vector Laboratories) and counterstained with hematoxylin (Sigma-Aldrich). The percentage score was defined as follows: 0: 0–5%; 1: 5–25%; 2:, 26–50%; 3: 51–75%; 4: 76–100%; and the staining intensity was defined as 1: weak; 2: moderate; and 3: strong and provided a Multiple index (MI) score (MI = intensity × percentage) as follows: MI = 0, scored as 1; MI = 1–4, scored as 2; MI = 5–8, scored as 3; MI = 9 or 12, scored as 4. Samples with scores ≥ 3 were considered to exhibit high expression, whereas those with scores ≤ 2 were classified as exhibiting low expression.

Paraffin sections on Superfrost Ultra Plus Adhesion Slides (Thermo Fisher Scientific Inc, Waltham, MA, USA) were deparaffinized and rehydrated in ethanol. Fibronectin immunohistochemistry was performed with rabbit polyclonal anti-fibronectin antibody (1:2000, Sigma-Aldrich, Budapest, Hungary), using the avidin–biotin method [30 (link)]. HNE and NT immunohistochemistry was performed with mouse monoclonal antibody (HNE clone: HNEJ-2, JaICA, Japan; NT clone: #189542, Cayman Chemical Company, Michigan, IL). Color development was induced by incubation with diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA). Pictures were taken from the stained sections for further analysis. The fibronectin stained area was quantified with Image J software.

Immunostaining was performed on mice tissue sections that had been confirmed as bladder cancer beforehand by a pathologist. The sections were deparaffinized in xylene, rehydrated with ethanol, and then blocked with 3% H₂O₂ followed by blocking in goat serum and incubation with anti-EZH2 antibody overnight at 4 °C. Secondary staining was performed and colorized using Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). The peroxidase reaction was developed with diaminobenzidine (DAB kit; Vector Laboratories) and the slides were counterstained with hematoxylin. The images were observed using a microscope (Olympus BX60, Tokyo, Japan) and quantitatively analyzed by Image-Pro Plus software (Media Cybernetics, Carlsbad, CA, USA).

Immunohistochemistry was performed with paraffin-embedded sections using standard protocols. Briefly, liver sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Endogenous peroxidase was blocked with 3% hydrogen peroxide (H₂O₂). The sections were boiled in 10 mM citrate buffer (pH 6.0) for 15 min for antigen retrieval. After blocking with 5% normal goat serum, sections were incubated at 4°C overnight with the following primary antibodies: anti-CCL2 (1:100; ab7814, Abcam, Cambridge, MA, USA), anti-CXCL9 (1:50; bs-2551R, Bioss, Beijing, China), and anti-IRF7 (1:250; bs-2994R, Bioss). Thereafter, the sections were rinsed in PBS and incubated with a biotinylated secondary antibody. The antigen was visualized with a diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin. A negative control without the primary antibody was included. Images were acquired using a Nikon Eclipse 80i fluorescence microscope and a DS-Fil CCD camera (Nikon).

To verify the results of this study, we collected 54 paired tumor tissues and adjacent tissues from our hospital to carry out immunohistochemistry. Immunohistochemistry was performed as described previously (24 (link), 25 (link)) using anti-AKR1C1(ab192785, Abcam) and anti-CARS1(ab126714, Abcam) antibodies. In brief, colon cancer tissues and adjacent tissues sections were deparaffinized in xylene and rehydrated with ethanol. Heat mediated antigen retrieval was performed in 10 mM citrate buffer at pH 6.0. After blocking with normal goat serum, slides were incubated with primary antibodies against AKR1C1 and CARS1 overnight (4°C). Tissue sections were then stained with biotinylated secondary antibody (Vector lab) for 1 h at room temperature, followed by the Vectastain Elite ABC reagent (Vector lab) for 30 min. The peroxidase reaction was developed with diaminobenzidine (DAB kit; Vector lab) and the slides were counterstained with hematoxylin (Sigma).
The positive expression of AKR1C1 and CARS1 were mainly located in the cytoplasm. Positive cells accounted for a percentage score standard: 0 = no positive cells; 1 = 1–25% positive cells; 2 = 26–50% positive cells; 3 = 51–75% positive cells; 4 = more than 75% positive cells.