Diaminobenzidine tetrachloride dab

For one case (S1), visualization of the CTB was enhanced using immunohistochemistry. Sections were washed in PBS containing 0.3% Triton X-100 (TX-100) three times for 5 min, quenched in 0.6% peroxide and PBS solution for 10 min, and placed in blocking solution with agitation for 1 h. Blocking solution consisted of 2% bovine serum albumin, 3.75% normal goat serum, 0.3% TX-100, all in PBS. Primary antibody (anti-CTB, described in Table 2) was added to the blocking solution and the tissue was incubated for an additional 48 h at 4°C. The sections were washed five times for 5 min, then placed in secondary antibody solution (Biotin-SP conjugated donkey anti-goat, described in Table 2) and incubated with agitation for 90 min at room temperature. These sections were washed five times for 5 min in PBS, placed in ABC solution (Vector Labs) for 90 min with agitation, washed five times for 5 min, and placed in diaminobenzidine tetrachloride (DAB, Sigma Aldrich, St. Louis, MO, USA) solution for 10 min with agitation. Then 0.025% peroxide was added and the sections were allowed to stain for approximately 3.5 min until there was visible contrast in the tissue. The sections were then washed with PBS five times, mounted on gelatin-coated slides, air-dried and dehydrated through ascending concentrations of ethanol before being cleared in xylenes and coverslipped with DPX.

Monokaryons were grown on MESM Petri dishes until they reached a diameter of 30 ± 3 mm. For superoxide radical (O2⁻) detection, the colonies were flooded with 10 mL of staining solution containing 5 mM 3-(N-morpholino) propane sulfonate NaOH buffer, pH 7.6, and 2.5 mM of Nitro Blue Tetrazolium (NBT, Sigma-Aldrich, St. Louis, MO, USA) and incubated at 24 °C for 30 min. The staining solution was discarded, and plates were incubated for 1.5 h at 24 °C in the dark.
For H2O2 detection, a similar procedure to the NBT was applied using a staining solution of 100 mM potassium phosphate buffer, pH 6.9, 2.5 mM Diaminobenzidine tetrachloride (DAB, Sigma-Aldrich), and 5 purpurogallin units/mL of horseradish peroxidase (Type VI, Sigma-Aldrich) freshly prepared and shielded from light to avoid spontaneous photo-oxidation. The reaction produced a brownish polymer in the mycelium.

To determine the cellular localization of Cyp17a2 in the testis and head-kidney of tilapia, the testes and head-kidneys from wild-type males at different development stages were dissected and fixed in Bouin’s solution for 12 hours at room temperature, then dehydrated and embedded in paraffin. The tissue blocks were then sliced into 5-μm-thickness sections, then the sections were treated in a blocking solution (5% BSA diluted in 1x PBS) (Sangon Biotech, China), incubated with the primary antibody against Cyp17a2 overnight at 4°C and rinsed with 1x PBS five times for 5 min per wash. In this study, the anti-Cyp17a2 polyclonal antibody was diluted at 1:1000 before use. As a negative control, the primary antibody was replaced with normal rabbit serum. The slides were then incubated with anti-rabbit immunoglobulin G (diluted at 1:1000) at room temperature for 1 h, and then rinsed with 1x PBS three times for 5 min per wash. Immunoreactive signals were visualized using diaminobenzidine tetrachloride (DAB) (Sigma, Germany) as the substrate. Sections were then counterstained with hematoxylin. Finally, all the images for these sections were acquired with an Olympus BX5 light microscope (Olympus, Japan).