After hypoxia induction and treatment, the cells were washed twice with 200 μL of PBS. Cells were incubated for 15 minutes with a solution of 5 mL of DAPI dye (Solarbio, Beijing, China) in 500 mL PBS. Cells were incubated for 15 min in the dark, washed twice with PBS, and then observed using light microscopy (U-RFLT50, Olympus, Japan).
Dapi dye
Manufactured by Solarbio
Sourced in China
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is a fluorescent dye used in molecular biology and microscopy applications. It binds strongly to adenine-thymine (A-T) rich regions in DNA, emitting blue fluorescence upon excitation. DAPI is commonly used for nuclear staining and DNA visualization in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
U rflt50, by Olympus (1 mentions)
Prim 1640, by Thermo Fisher Scientific (1 mentions)
2 methylimidazole, by Energy Chemical (1 mentions)
Zncl2, by Maclin Biochemical Technology (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Nanozoomer s60, by Hamamatsu Photonics (1 mentions)
Rabbit anti rat p2x7 antibody, by Alomone (1 mentions)
Laser confocal microscope, by Olympus (1 mentions)
4 protocols using dapi dye
1
DAPI Nuclear Staining Protocol
2
Synthesis and Characterization of Zinc-based Nanoparticles
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2-methylimidazole (98%) was purchased from Energy Chemical Co., Ltd (Shanghai, China). ZnCl2 was purchased from Macklin Biochemical Co., Ltd (Shanghai, China). Cetyltrimethylammonium bromide (CTAB) was purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Sodium ethylene diamine tetraacetic acid (EDTA-Na2) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Nucleic acid dye SYBR GREEN II, 4% tissue fix solution, and DAPI dye were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The Formvar support film was purchased from Zhongjingkeyi Film Technology Co., Ltd. (Beijing, China). The cell Counting Kit-8 (CCK-8) kit was purchased from LABLEAD. Inc (Beijing, China). 3′,6′-Dihydroxy-5-isothiocyanato-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one (FITC), PRIM-1640, 0.25% trypsin–EDTA (1×), PBS (1×), penicillin–streptomycin solution and LysoTracker™ Red DND-99 lysosomal probe were purchased from Thermo Fisher Scientific (Massachusetts, USA). Dendritic cells 2.4 were saved by our laboratory. All other reagents were all analytical grade and used without further treatment. Unless otherwise specified, deionized water for injection was used for solution preparation.
3
Apoptosis Detection in Colon Sections
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TUNEL staining was performed using paraffin-embedded tissues as mentioned before. The staining was performed with apoptosis kit (11684817910, Roche) to detect apoptotic cells in colon sections according to the instructions of manufacturer. After the paraffin sections conventional dewaxing to water, performed the antigen recovery with protease K working solution at 37°C for 22 min. Then incubated the sections with permeabilize working solution at room temperature for 20 min. Next, take appropriate amount of TDT enzyme, dUTP and buffer in the tunel kit according to the number of slices and tissue size and mix at 1:5:50 ratio, and used to incubate the tissues at 37°C for 1 h. Finally, the DAPI dye (S2110, Solarbio) was used to stain nucleus at room temperature for 10 min. Images were captured with NanoZoomer S60 (C13210-01, Hamamatsu).
4
Immunofluorescence Analysis of P2X7 and GFAP in Rat Trigeminal Ganglia
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Rats were anesthetized with 10% chloral hydrate normal saline solution (0.35 mL/100 g). On the 14th day after operation, normal saline and fresh 4% paraformaldehyde were perfused through the heart. Then, we removed TG, fixed it with 4% paraformaldehyde for 1 day, then transferred it to 20% sucrose solution for one day, and finally soaked for one day with 30% sucrose solution. Then, TG sections (10 µm) were prepared on a freezing microtome and placed on immunosorbent slides. The sections were washed, three times, with PBS buffer, sealed with 5% BSA at 37 °C for 30 min, and incubated overnight with rabbit anti-rat P2X7 antibody (1:8000, Alomone Labs, Jerusalem, Israel) and mouse anti-rat GFAP antibody (1:1000, Boster) in a wet box at 4 °C [28 (link),30 (link)]. After 12 h, the slide was removed and washed, 3 times, with PBS buffer, then, the tissue sections were incubated in CY-3 goat anti-rabbit antibody (1:200, Boster, Wuhan, China) and FITC goat anti-mouse antibody (1:200, Boster) in a dark environment at room temperature for 1 h. Next, the sections were washed again, 3 times, with PBS buffer and sealed with anti-fluorescence quenching sealing tablets containing DAPI dye (Solarbio, Beijing, China). The photographs of the samples were taken with a laser confocal microscope (Olympus, Tokyo, Japan).
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