Denuded oocytes (DOs) were obtained by transferring COCs in 300 μL of 10 mg/mL hyaluronidase solution for 15 min. Then DOs were incubated in the fixation solution (4% paraformaldehyde/PBS) for 30 min, in the permeabilization solution (1% Triton X-100/PBS) for 1 h, and in the blocking solution (1% BSA-supplemented PBS) for 1 h at room temperature (RT), followed by incubation with α-tubulin-FITC antibody (1:200), acetyl-α-tubulin antibody (1:100), γH2A.X antibody (1:100) or phalloidin-TRITC (1:100; Sigma-Aldrich; Cat# P1951) overnight at 4°C. After washes in PBST, oocytes were incubated with the corresponding secondary antibodies for 1 h and counterstained with 10 μg/mL Hoechst 33342 or propidium iodide (PI) for 10 min at RT. In addition, oocytes were stained at 38.5°C for 30 min with 500 nM MitoTracker Red CMXRos (Thermo Fisher Scientific; Cat# M7512) for mitochondrion staining, with 2 μM MitoProbe JC-1 (Thermo Fisher Scientific; Cat# M34152) for mitochondrial membrane potential assessment, with 10 μM dichlorofluorescein diacetate (DCFHDA; Beyotime, Huangzhou, China; Cat# S0033S) for ROS staining, and with Annexin-V-FITC (1:10; Beyotime; Cat# C1062) for apoptosis assessment. Lastly, oocytes were mounted on the glass slides and imaged under a confocal microscope (LSM 700 META, Zeiss, Germany).
Dichlorofluorescein diacetate dcfh da
Manufactured by Beyotime
Sourced in China
Dichlorofluorescein diacetate (DCFH-DA) is a cell-permeable fluorogenic probe that is commonly used to measure oxidative stress in biological systems. It is non-fluorescent until it is hydrolyzed by intracellular esterases and then oxidized by reactive oxygen species (ROS), resulting in the formation of the highly fluorescent 2',7'-dichlorofluorescein (DCF).
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Lsm 700 meta, by Zeiss (1 mentions)
Annexin 5 fitc, by Beyotime (1 mentions)
Mitoprobe jc 1, by Thermo Fisher Scientific (1 mentions)
K sfm medium, by Thermo Fisher Scientific (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Edta solution, by Thermo Fisher Scientific (1 mentions)
Temozolomide, by MedChemExpress (1 mentions)
4 protocols using dichlorofluorescein diacetate dcfh da
1
Oocyte Immunostaining and Mitochondrial Analysis
2
Isolation and Culture of Primary Murine Keratinocytes
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Primary keratinocytes were obtained from skin of newborn BALB/c mice (postnatal day 1–3) as described previously29 (link). Briefly, the keratinocytes was isolated from the skin by 0.25% trypsin/0.04% EDTA solution (Invitrogen, USA) at 4 °C overnight. The keratinocytes were then plated into dishes that were pre-coated with fibronectin (40 μg/mL) and cultured in serum-free medium (K-SFM medium) (Gibco, USA) supplemented with 100 U/ml penicillin (Invitrogen, USA), and 100 mg/ml streptomycin (Invitrogen, USA). The cells were incubated at 37 °C in 5% CO2 and 95% humidity for 24 hours and washed gently with warm phosphate-buffered saline (PBS) to discard non-adherent cells. The media were then refreshed. N-acetyl-l-cysteine (NAC) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE) were purchased from Beyotime (cat. no. S0063, Haimen, China).
3
Temozolomide and Ferrostatin-1 Protocol
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Temozolomide (purity>99%) and ferrostatin-1 (Fer-1) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Dichlorofluorescein diacetate (DCFH-DA) was purchased from the Beyotime Institute of Biotechnology (Beijing, China).
4
Histological Analysis of Mouse and Human Livers
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Mouse livers were dissected and fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. Livers were embedded in paraffin and sectioned at 6 μm. Histology and immunohistochemistry were performed following standard procedures. For Oil Red O staining, liver tissues were fixed in 4% PFA overnight and frozen in OCT compounds. The sections were cut at 10 μm and allowed to air dry. Then the slides were briefly washed with running tap water and rinsed with 60% isopropyl alcohol. The sections were stained with freshly prepared Oil Red O solution (0.3% Oil Red O in 60% isopropyl alcohol) for 15 min and rinsed with 60% isopropyl alcohol followed by lightly staining nuclei with hematoxylin. Human fatty liver samples were purchased from Fanpu Biotech, Inc. (Guilin, China). For ROS staining, mouse livers were fixed in 4% PFA overnight and embedded in OCT compounds, sections were cut at 10 μm. Sections were incubated with dichlorofluorescein diacetate (DCFH-DA) (Beyotime, China) for 30 min at 37 °C. Nuclei were stained with DAPI. For PAS staining, paraffin sections were oxidized in 0.5% periodic acid solution for 5 min, rinsed in distilled water, placed in Schiff reagent for 15 min, then washed in tap water for 5 min and counterstained in Mayer's hematoxylin for 1 min.
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