Immpress universal reagent

Two slices from three saline- and four PCP-treated animals per genotype (P2rx7^+/+ and P2rx7^−/−) were incubated for 10 min in 3% H₂O₂ in PB, rinsed three times for 10 min in PB, and three times for 10 min in PB. The pre-made blocking buffer (ImmPRESS UNIVERSAL REAGENT, Vector Laboratories, MP-7500) was supplemented with 0.5% Triton X-100 (Tx, Merck-Sigma), and 7.5% of normal donkey serum (NDS, Jackson Immunoresearch, Europe). After 1 h of blocking, slices were incubated with the primary rabbit anti-c-FOS antibody (1:1,000, Santa Cruz Biotechnology sc-52, Dallas, TX, USA) in PB containing 0.05% sodium azide (Merck-Sigma) for 24 h at room temperature (RT) and 72 h at 4°C. Eventually, the slices were rinsed in PB and incubated with the premade secondary anti-mouse/rabbit HRP-conjugated ImmPRESS UNIVERSAL REAGENT (Vector Laboratories, MP-7500) for 1 h. DAB was developed using the commercially available DAB-REACTION KIT (Vector Laboratories, SK-4105) according to the manufacturer’s instructions.

Both juvenile and adult fish were sacrificed with an overdose of benzocaine (0.2 g/L). Gonad samples were dissected, fixed in Bouinʼs solution overnight at room temperature, and stored in ethanol 70% until further processing. Samples were dehydrated in ascending ethanol series, embedded in paraffin, sectioned transversally at 5 µm thickness, and stained with hematoxylin–eosin (HE). For immunohistochemistry analysis sections were deparaffined, re-hydrated, and submitted to antigen retrieval with citrate buffer (pH 6.0) in microwave for 10 min. Endogenous peroxidase activity was blocked using 3% H₂O₂ solution in 0.1 M PBS at room temperature for 30 min. Sections were rinsed in 0.1 M PBS and blocked with horse serum from ImmPRESS Universal reagent (Vector Laboratories). Sections were incubated with the primary antibody #189, specific for undifferentiated spermatogonia (1:500 dilution)^{44 (link)}, for 16 h at 4 °C, rinsed in 0.1 M PBS, and incubated with ImmPRESS Universal secondary antibody (Vector Laboratories) for 30 min at RT. Then, sections were rinsed in 0.1 M PBS, incubated with ImmPACT DAB HRP substrate (Vector Laboratories), counter-stained with hematoxylin and mounted.

After processing and inclusion of samples in paraffin, histological sections with a thickness of 4 mm were used to study the immunohistochemistry. The sections were deparaffinized through a series of “baths” in xylene and alcohol. With the aim of improving the responses, one treatment stage of the blades was performed with citrate buffer. Endogenous peroxidase was blocked using a solution composed of methanol and hydrogen peroxide (30%). Antibodies were used specific for macrophages CD 68 (AbD Serotec), being a marker of inflammatory response in tissue. A biotinylated anti-mouse secondary antibody was used (ImmPRESS™ Universal Reagent, Vector Laboratories). Subsequently, we used the avidin-biotin complex. The color development occurred by adding the chromogen 3,3-diaminobenzidine (DAB, 3,3-diaminobenzidine, Easypath®) on the cuts by the reaction thereof with the avidin-biotin-peroxidase complex. The counterstaining was conducted by Harris hematoxylin bath. Twelve microscopic fields were analyzed with 40x magnification for counting the labeled cells in the ImageJ program.