Diaminobenzidine dab chromogen

Immunohistochemistry was performed according to the method described by Camargo et al. [35 (link)]. The antibodies used for the markers and dilutions in this study are listed in Table 3. Digestion of the antigen at high temperature was performed in a pressure cooker for 1 min using citrate buffer (6.0 pH). For incubation with the primary antibody, the sections were diluted in bovine serum albumin (BSA) solution and applied to each slide. Then, the slides were incubated overnight in a humid chamber at 4 °C for 18–22 h. The slides were then washed in phosphate-buffered saline (PBS) and incubated with the secondary antibody using the ABC Kit with Vectastain (Vector Laboratories, CA, USA) (Table 3). To visualize the positive cells, the slides were washed in PBS and proteins were visualized using diaminobenzidine (DAB) chromogen (70 mg DAB in 110 mL of the Tris-HCl; Sigma Chemical Co., St. Louis, MO, USA) and Harris hematoxylin (Merck, Darmstadt, Germany), and finally mounted with microscopy resin.

An immunohistochemistry protocol was performed on the semen smears to detect immunohistochemical expressions and the immune location of AQP-3 (Thermo Fisher Scientific USA), AQP-7 (Protein tech USA), and AQP-8 (Abcam UK) molecules in sperm cells. In brief, the slides were washed in PBS and incubated for 15 min in 1% bovine serum albumin (BSA) for blocking. The BSA was removed without washing then primary antibodies of AQP-3 (1:100), AQP-7 (1:100), and AQP-8 (1:100) were dropped onto the samples and incubated at 4 °C in a humidified and dark chamber overnight. After washing with PBS for 5 min at room temperature, the samples were incubated with biotinylated secondary antibody (Vector Laboratories, USA) at room temperature in a humidified dark environment for 30 min and again washed with PBS incubated with horseradish peroxidase (HRP)-labeled streptavidin secondary antibody (GE Healthcare UK) at room temperature for 20 min. After washing with PBS, immune expressions were made visible adding diaminobenzidine (DAB) chromogen (Sigma, Germany) onto the samples. For the negative control, some slides were simultaneously treated with PBS instead of the primary antibodies. The immunochemical stained slides were mounted with a coverslip following counterstaining with hematoxylin and washing with distilled water.

Mice were sacrificed on the 24th day following the first injection of the test reagents, and tumors were removed. Tumors were fixed with formalin and embedded in paraffin. The 4 µm paraffin sections were stained by hematoxylin-eosin (HE, Beyotime).
For immunohistochemistry, 4 µm paraffin sections were incubated with each of primary antibodies (anti-Ki67, anti-CD31 and anti-caspase 3, Sigma-Aldrich) at 1:200 dilution overnight at 4 °C. The sections were washed three times with PBS for 5 min each and incubated with HRP-labeled goat anti-rat secondary antibody (Sigma-Aldrich). After the incubation for 1 h at 37 °C, the sections were washed three times with PBST for 5 min each and incubated with diaminobenzidine (DAB) chromogen (Sigma-Aldrich) for 3–5 min to show a dark brown color. The sections were photographed using an Olympus IX70 light microscope (Olympus, Tokyo, Japan), and the integrated optical density (IOD) of each image was analyzed with Image-Pro Plus analysis software (Media Cybernetics).