Chemidoc xrs chemiluminescence imaging system

Total RNA content was extracted from each cell line using the TRIzol reagent. The solid sample of RNA was dissolved in the loading buffer, denatured for 10 minutes at 65°C and subjected to 2% formaldehyde gel electrophoresis. The RNA in formaldehyde gel was transferred to a nylon membrane using the syphon method and allowed to stand overnight at room temperature. After being heated at 120°C for 1 hours, the membrane was hybridized and detected using the Dig Northern Start Kit, Dig High Prime DNA Labeling and Detection Starter Kit II. After that, the membrane was added with 2‐3 drops of cytidine 5′‐diphosphate, and the results were visualized using the ChemiDoc XRS + chemiluminescence imaging system (Bio‐Rad) for 30 seconds.

Western blot was performed as previously reported [13 ]. Briefly, the cells were harvested, washed and lysed with 1× RIPA buffer. Thermo BCA protein assay kit was used to test the protein concentration. Equal amounts of protein from cell lysates or animal tissue were solubilized in 5× SDS sample buffer and separated on 8–10% SDS polyacrylamide gels, and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk in TBST and incubated with primary antibodies against Caspase-3, Caspase9, Cyt-C, Bcl-2, Mcl-1, Bax, Bim and STAT3 proteins at 4 °C overnight. Then the membranes were washed and incubated with a secondary antibody against rabbit IgG for 1 h, followed by washing and transferring into ECL solution (Millipore, Darmstadt, Germany), and scanned using the Bio-Rad ChemiDoc XRS+ Chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). All the results were analyzed by Image J software.

Samples were homogenized with RIPA buffer (150 mM sodium chloride, Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) at 4°C for 30 min, then were centrifuged for 15 min at 4°C. BCA protein assay kit (Boster, Wuhan, China) was used to determine the protein levels in supernatant. The samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States). Bands were blocked with 5% BSA in TBST (0.1% Tween 20 in Tris-buffered saline) for 1 h at room temperature. Relative primary antibodies were incubated at 4°C overnight: rabbit Keap1 (1:1,000, Affinity, Cincinnati, OH, United States), rabbit Nrf2 (1:1,000; Affinity, Cincinnati, OH, United States), and mouse GAPDH (1:1,000, Qidongzi, Wuhan, China). Then bands were washed with TBST and incubated second antibody for 2 h at room temperature: goat anti-rabbit IgG horseradish peroxidase or goat anti-mouse IgG horseradish peroxidase (1:5,000, Qidongzi, Wuhan, China). Finally, these bands were detected by enhanced chemiluminescence reagents (Qidongzi, Wuhan, China) with the ChemiDoc XRS chemiluminescence imaging system (Bio-Rad, Hercules, CA, United States).

Western blot analysis was performed as previously described.^{43 (link)} Briefly, whole-cell lysates were prepared in RIPA lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris, 5 mM NaF, and 0.1% SDS) and aliquots of 30 μg protein were separated by SDS-PAGE and blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). Membranes were blocked at room temperature For 1 h in 5% non-fat powdered milk in Tris-buffered saline, followed by an overnight incubation at 4 °C with specific antibodies. After incubation with appropriate HRP-conjugated secondary antibodies (Santa Cruz), blots were developed using an enhanced chemiluminescence (ECL Kit, Millipore) and exposed in ChemiDoc XRS chemiluminescence imaging system (Bio-Rad). Antibodies used in this study are listed in supplementary Table 1.

The proteins were extracted from ovaries obtained from the CON group, the VCD group, and the DBT group and were boiled with proper loading buffer for 10 minutes. Protein concentration was measured by BCA assay. Fifty micrograms of protein from each group was separated by 12% SDS-PAGE and then transferred onto a nitrocellulose filter membrane. The membranes were blocked with 5% BSA for 1.5 h at room temperature and then incubated with the appropriate primary antibody at 4°C overnight. The next day, the membranes were washed with TBST 3 times (10 minutes each time). The membranes were incubated with the corresponding secondary antibodies for one hour. After 3 washes with TBST, blot images were obtained with a Bio-Rad ChemiDoc XRS + chemiluminescence imaging system. The results were analyzed by ImageJ software.

A carbon dioxide incubator was produced by Thermo Fisher Scientific Inc., USA. An HM340E-rotary slicer and a hematoxylin–eosin (HE) staining fully automatic dyeing machine were produced by MICROM Inc., Germany. A vertical electrophoresis apparatus and transmembrane system were produced by Bio-Rad Inc., Hercules, California, USA. The ChemiDoc™ XRS + chemiluminescence imaging system was also produced by Bio-Rad Inc. The BD FACSCalibur™ flow cytometer was produced by BD biosciences Inc., California, USA. An inverted fluorescence microscope was produced by Carl Zeiss Co. Ltd., Germany.

Fifty milligrams of lung tissue were homogenized with RIPA lysis buffer (containing phosphatase inhibitor). The protein content of lung tissue was quantified, and then the separation gel and concentrated gel were prepared based on the molecular weight, and a suitable sample was taken for loading. Subsequently, electrophoresis and membrane transfer were performed. The PVDF membrane was removed and blocked with 5% skim milk for 2 h, combined with the primary antibody overnight at 4 °C, and incubated with the secondary antibody for 2 h. The membrane was washed 3 times with TBST for 5 min each time and the immunoreactivity bands were detected by ChemiDoc XRS + chemiluminescence imaging system (Bio-Rad, USA).