Sf9 insect cells were grown on coverslips in 35 mm plates and transfected with the virus of the WT or mutants of ABCG25. Forty-eight hours after transfection, cells were fixed in 4% (w/v) paraformaldehyde for 30 min at 4 °C, subsequently permeabilized and blocked with PBS containing 0.3% (w/v) Triton X-100 and 3% (w/v) bovine serum albumin for 1 h at room temperature. For immunostaining, samples were incubated with the anti-Flag tag (DYKDDDDK) Alexa Fluor 594-conjugated antibody (1:50, CST) for 1 h at 37 °C, followed by incubation with 5 μM Dio (Absin) for 20 min at 37 °C. Nuclei were visualized with Hoechst 33342 (LEAGENE). All fluorescence images were captured via the LSM880 confocal laser scanning microscope (Zeiss) and analysed with ZEN (Zeiss).
Hoechst 33342
Manufactured by Leagene
Sourced in China
Hoechst 33342 is a fluorescent dye that binds to the minor groove of double-stranded DNA. It can be used for DNA staining and nuclear counterstaining in various applications, such as flow cytometry and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Methacrylic anhydride, by Merck Group (1 mentions)
Isophorone diisocyanate, by Merck Group (1 mentions)
Type a, by Merck Group (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Gelatin, by Merck Group (1 mentions)
96 well glass bottom dishes, by Cellvis (1 mentions)
3 protocols using hoechst 33342
1
Visualizing ABCG25 Protein Localization
2
Multifunctional Hydrogel for Biomedical Applications
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Polyethylene glycol (PEG, Mn = 400), l -Arginine (L-Arg), β-Cyclodextrin (β-CD) were obtained from China Sinopharm Group Reagent Co, Ltd. Isophorone diisocyanate (IPDI), Gelatin, Type A and Methacrylic anhydride (MA) were obtained from Sigma-Aldrich (Shanghai, China). Stannous caprylate, dimethylol propionic acid (DMPA) were bought from Alfa Aesar Corporation (Shanghai, China). Other raw materials such as acetone and triethyl amine (TEA) were obtained from Kelong Chemical Corporation (Chengdu, China), and used without further purification. Hoechst33342 was purchased from Leagene Biotech Co., Ltd. (Beijing, China). The Calcein AM was obtained from Shanghai yaji biotechnology Co., Ltd. (Shanghai, China). The CCK-8 kit was obtained from Dojindo (Japan).
3
URAT1 Protein Localization Assay
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The wild-type or mutated URAT1 genes were cloned and inserted into the pCDNA3.1 vector with an N-terminal FLAG and a C-terminal mRuby3 tag, respectively. When the cell density reached 1.0×10 6 cells/mL, the HEK293F cells were transiently transfected with the indicated plasmids and incubated for an additional 24 h. The HEK293F cells were seeded in 96-well glass bottom dishes (Cellvis) at a density of 1.0×10 5 cells/well. The cell nuclei were stained with Hoechst 33342 (LEAGENE, 1210A21), and fluorescence images were acquired using a high-resolution laser confocal system (Leica STED). Cell lysates were subjected to immunoblotting with an anti-Flag antibody (Abmart, M20026H) to detect the expression levels of URAT1 variants.
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