Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody

After treatment with various doses of tryptamine, PC-3 cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 mM PMSF, and the total protein concentration was determined by BCA Protein Assay Kit (Solarbio, Beijing, China). Samples were electrophoresed on a 10% sodium dodecylsulfate (SDS) polyacrylamide gel and electrotransferred onto polyvinylidene difluoride (PVDF) membrane (Beyotime, China). After blocking with 5% BSA for 1 h, the membrane was incubated with the primary antibodies rabbit anti-cleaved caspase-3 (1:1000, ab32042, Abcam) and anti-β-actin (1:1000, 4967S, Cell Signaling Technology, Boston, MA, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000, 7074S, Cell Signaling Technology) for 1 h at 37 °C. After washing with TBST, the bands were visualized by the enhanced chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA) and Odyssey^® imaging system (LI-COR, Lincoln, NE, USA) according to the manufacturers’ instructions.

The expression of both TNFα and IL10 proteins were detected in liver tissues of all groups using western blot assay. This assay was done as previously described^{66 (link)}. Briefly, liver specimens were lysed using RIPA lysis buffer. The obtained proteins were separated on SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (PVDF). PVDF membranes were separately incubated with the following mouse monoclonal primary antibodies: anti-TNFα (1:100 dilution, Santa Cruz Biotechnology, Cat # sc-52746), anti-IL10 (1:200 dilution, Santa Cruz Biotechnology, Cat # sc-365858), and anti-β-actin (housekeeping, 1:200 dilution, Cat # sc-47778). PVDF membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000 dilution, Cell Signaling Technology, Beverly, MA, USA). The color was developed using tetramethylbenzidine (Sigma) and the band density was quantified by ImageJ software.

Frozen testis tissue was homogenized in ice-cold radioimmunoprecipitation assay buffer (pH 7.5) containing protease inhibitor cocktail (Roche, USA). After denaturation at 95°C for 5 min, samples were centrifuged at 12,000 × g for 20 min at 4°C and the supernatant was collected and stored at −20°C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane that was blocked overnight at 4°C in PBS containing 0.1% Tween 20 (PBS-T) and 5% skim milk, and then probed with rabbit anti-rat antibodies against the following proteins: Bcl-2 (1:1000; ab59348) and Bax (1:1000; ab32503) (both from Abcam, USA); cytochrome C (1:500; sc13156) (Santa Cruz Biotechnology, USA); cleaved caspase-9 (1:1000: #9506) and cleaved caspase-3 (1:500; #9664) (both from Cell Signaling Technology, USA); and β-tubulin (1:5000; YM3030) (Immunoway, USA). After three washes with TBS-T, the membrane was incubated at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000; Cell Signaling Technology) in PBS-T with 2% skim milk and washed three times with TBS-T [35 (link), 36 (link)]. Immunoreactivity was visualized by enhanced chemiluminescence. The relative signal intensity of protein bands was analyzed with Image J software (National Institutes of Health, USA).

Equal quantities of protein from the testis tissue lysate were processed for Western blotting (Roche, USA). Each sample was denatured, electrophoresed, and transferred onto a polyvinylidenedifluoride membrane. Specific steps were as follows: proteins were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis after denaturation at 95 °C for 5 min, and transferred to a polyvinylidenedifluoride membrane that was blocked overnight at 4 °C in PBS containing 0.1% Tween 20 (PBS-T) and 5% skim milk. The primary antibody used was rabbit anti-rat TSSK2 (1:500, Abcam, USA). After three washes with TBS-T, the membrane was incubated at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:1000; Cell Signaling Technology) in PBS-T with 2% skim milk and washed three times with TBS-T. The blots were visualized with LumiGLO reagent and peroxide, followed by exposure to X-ray film. Western blot analyses were performed at least in triplicate.