Tunel staining kit

Apoptosis of pancreatic islets was evaluated using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining kit (Chemicon International, Temecula, CA, USA). Microphotographs of TUNEL-stained islets were taken using an Olympus BX53, DP 73 digital camera and DP Controller software (Olympus, Tokyo, Japan).

The Achilles tendon along with part of the tibia and calcaneus were taken and quickly placed in 4% paraformaldehyde for fixation. The fixed specimens were routinely sectioned and stained. Masson staining was used to image the collagen fiber using a commercial kit (Masson's Trichrome Stain Kit, Solarbio, Beijing, China) according to the manufacturer's instructions. Paraffin-embedded tissue sections were then deparaffinized in xylene and dehydrated in ethanol gradients. The staining was performed according to the instructions of the TUNEL staining kit (Sigma‒Aldrich, USA). Apoptotic cells were positive cells, which were brownish yellow or tan under the light microscope, and non-apoptotic cells were negative cells, which appeared blue.

Oxymatrine (purity > 98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Malondialdehyde (MDA) (cat.no. S0131M), glutathione peroxidase (GSH-Px)(cat.no. S0059S), superoxide dismutase (SOD)(cat.no. S0087), and catalase (CAT)(cat.no. S0082) were purchased from Beyotime (Shanghai, China). IL-6 and NF-κB (p65) were obtained from ELISA kits (R&D Systems Inc., Minneapolis, MN, USA; and MyBioSource, Southern California, San Diego, USA). Secondary antibodies, such as Alexa Fluor®488 labeled and Alexa Fluor®594 labeled goat anti-rabbit IgG (H + L), primary antibodies pSTAT3 (ab76315) were obtained from Abcam (Cambridge, UK). The primary antibodies STAT3 (bs-1658R and bsm-52235R) was purchased from Bioss. anti-Janus kinase 2(JAK2) (cat.no. 3230) and pJAK2 (cat.no. 3771) were obtained from Cell Signaling Technology. TUNEL staining kit was obtained from Sigma-Aldrich Co., Ltd.

RPMI-1640 medium and fetal bovine serum (FBS) were acquired from Gibco BRL (Gaithersburg, MD, USA). RA was purchased from the China National Institute for the Control of Pharmaceuticals, dissolved in dimethyl sulfoxide (DMSO), and stored at −20°C. MTT, TUNEL staining kit, and LY294002 were obtained from Sigma Chemical Company (St. Louis, MO, USA). Annexin-V/Propidium Iodide (PI) apoptosis detection kits were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Primescript reverse transcription reagent kits with gDNA erasers were obtained from TaKaRa (Dalian, China). TRIzol reagent and Power SYBR Green PCR Master Mixes were purchased from Life Technologies (Grand Island, NY, USA). The primary antibodies against BAX, Bcl-2, PARP, caspase-3, cleaved caspase-3, cyclinD1, CDK2, PI3K, AKT, p-PI3K, p-AKT, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). Fluorescein-conjugated secondary antibodies were obtained from Odyssey (Licor, Belfast, ME, USA). The mice were obtained from Kevins Animal Company (Changzhou, China) and maintained in a specific-pathogen-free (SPF) environment with adequate food and water.

Cells were exposed to 25-μM purpurin or 200-mM DMSO immediately after treatment with 1-mM H₂O₂. For reactive oxygen species (ROS) formation, 20-μM 2′,7′-dichlorofluorescein diacetate (DCF-DA, Invitrogen Molecular Probes, Eugene, OR, USA) was added to HT22 cells at 10 min after H₂O₂ treatment to induce the formation of DCF, which has strong fluorescence. Cells were harvested 30 min after DCF-DA treatment. DNA fragmentation was validated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as described in previous studies [22 , 23 (link)]. Briefly, cells were harvested at 3 h after H₂O₂ treatment, and DNA fragmentation was visualized using a TUNEL staining kit (Sigma). Microphotographs from DCF-DA and TUNEL staining were taken using a confocal fluorescence microscope (LSM 510 META NLO; Zeiss GmbH, Jena, Germany), and the fluorescence intensity was measured using a Fluoroskan ELISA plate reader (Labsystems Multiskan MCC/340). Cell death was assessed using a WST-1 assay at 5 h after H₂O₂ treatment, and formazan fluorescence was measured using a Fluoroskan ELISA plate reader.

Oxidative stress in HT22 cells was induced by exposure to 1 mM H₂O₂, and 50 μg/mL CVE was added to HT22 cells simultaneously with H₂O₂. Ten minutes after H₂O₂ and CVE treatment, HT22 cells were incubated with 20 μM DCF diacetate (DCF-DA) to convert DCF-DA to DCF. To measure ROS formation, cells were fixed for 3 h after H₂O₂ and CVE treatment. Cells were stained with a TUNEL staining kit (Sigma-Aldrich, St. Louis, MO, USA) to detect DNA fragmentation induced by H₂O₂. DCF- and TUNEL-positive cells were observed by confocal fluorescence microscopy using an LSM 510 META NLO microscope (Carl Zeiss GmbH, Jena, Germany). DCF and TUNEL fluorescence intensities were measured using a Fluoroskan ELISA plate reader (Labsystems Multiskan). Cell viability was measured using the WST-1 assay 5 h after H₂O₂ and CVE treatment, as described above. In addition, apoptosis and anti-apoptotic factors, including Bax and Bcl-2, were assayed by western blot. Briefly, cells were harvested 6 h after H₂O₂ treatment and lysed with ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting for Bax and Bcl-2 was performed as described previously [45 (link)]. Antibodies to Bax, Bcl-2, and β-actin were purchased from Abcam (Cambridge, UK) and were used at 1:2000 dilutions.