One step tunel apoptosis assay kit

TUNEL assay was performed with One Step TUNEL Apoptosis Assay Kit (Meilunbio, MA0223) following the manufacturer’s instructions using testes and ovaries cryosections.

After being fixed with 4% paraformaldehyde for 10 minutes, and incubated in 0.3% Triton X‐100 solution for 10 minutes, brain sections underwent antigen retrieval by citrate antigen retrieval solution and blocked with 1% bovine serum albumin (BSA) for 1 hour. Then, the sections were incubated with primary antibodies anti‐P2Y6 receptor (1:50, Alomone), Iba1 (1:200, Novusbio), GFAP (1:200, Millipore), Tuj‐1 (1:200, Millipore), NeuN (1:200, Millipore), or Lamp‐2 (1:200, Millipore) overnight at 4°C. After three five minutes times washes in PBS, sections were incubated with fluorescent conjugated secondary antibodies for 1 hour at 37°C. The sections were observed under a TCS SP5 Confocal Scanning System (Leica). TUNEL staining (TdT‐mediated dUTP Nick End Labeling) was performed for apoptotic analysis, by using One Step TUNEL Apoptosis Assay Kit (Meilun Biotechnology Co., Ltd).
Cells were treated with 100% methanol (chilled at −20°C) for 5 minutes. They were subsequently washed with ice‐cold PBS and incubated with 1% BSA in PBS for 60 minutes to block unspecific binding. Then, cells were incubated in the primary antibody anti‐MLCK (1:100, Abcam), Iba‐1 (1:200, Novusbio) overnight at 4°C. After three times washes by PBS, cells were incubated with the secondary antibody for 1 hour at 37°C in the dark. These cells were imaged with laser scanning confocal microscopy (Leica).

Major pelvic ganglia were dissected from naive rats and dissociated with collagenase type II (2 mg/ml) and bovine serum albumin (0.6 mg/ml) for 30 min. They were then cultured on poly-D-lysine and laminin-coated coverslips. The cell medium comprises neurobasal medium, B27 supplement, fetal bovine serum, glia-derived neurotrophic factor, antibiotic/antimycotic and L-glutamine. After 72 h, pelvic neurons were treated with acrolein (the CYP metabolite, 0, 25, 50, and 100 μM) for 6 h to confirm a stimulation concentration of acrolein. After immunofluorescent staining of neuron-specific class III beta-tubulin to identify neurons, apoptosis assay was conducted using the Meilun One Step TUNEL Apoptosis Assay Kit (FITC). Images were visualized with a laser scanning confocal microscope and captured by ZEN software.

The DMSO- and 0.5 μM BI 2536-treated cells were labelled with FITC using the One Step TUNEL Apoptosis Assay Kit (Meilunbio) according to the manufacturer’s recommendations. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Biotec). Immunofluorescent staining was observed using a fluorescent microscope (Olympus).

Paraffin-embedded heart sections were stained with the One Step TUNEL Apoptosis Assay Kit (Dalian Meilun Biotechnology, China) to evaluate the apoptosis of myocardial cells stimulated by DOX. The average apoptotic index was defined as the ratio of the number of TUNEL-positive myocytes relative to DAPI-stained myocytes from 6 random fields in the section.

After being dewaxed and rehydrated with xylene and gradient ethanol, 5 μm sections of paraffin embedded testis tissue were stained with hematoxylin and eosin (H&E) for morphological analysis. Immunohistochemistry was performed according to the methods previously described [12 (link)]. Leydig cells were identified by staining for 3β-hydroxysteroid dehydrogenase (3β-HSD) and the number of 3β-HSD-positive cells was counted in 25 fields randomly selected on slides in each group of mice. TUNEL staining was performed using one step TUNEL apoptosis assay kit (Meilunbio, China). To positively identify the apoptotic Leydig cells, the tissue sections were blocked with 10% goat serum and stained with 3β-HSD (1:50) for an additional 2 h at 37 °C after TUNEL staining. The sections were subsequently incubated with a goat anti-rabbit IgG Alexa fluor 647 conjugated antibody (1:100) for 1 h at 37 °C and observed using a laser confocal microscope.