Cell-Light™ EdU Apollo®488 In Vitro Imaging Kit (RiboBio) was used according to the manufacturer’s instructions. Cells were incubated with 50 μM EdU at room temperature for 2 h, then fixed with 4% paraformaldehyde for 15 min and soaked with 0.5% Triton X-100 for 20 min. Hoechst 33,342 stain was used for 15 min. Finally, the cells were observed under the Leica Sp5II-CLSM microscope.
Sp5ii clsm microscope
Manufactured by Leica
Sourced in Germany
The Leica SP5 II CLSM is a confocal laser scanning microscope designed for high-resolution imaging of biological samples. It features a modular design, allowing for customization to meet specific research needs. The microscope's core function is to provide detailed, optical sectioning of specimens through the use of laser excitation and confocal detection.
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Lab products found in correlation
49 6 diamidino 2 phenylindole dapi, by Beyotime (2 mentions)
Albumin bovine 5, by Solarbio (2 mentions)
Alexa fluor 568 goat anti mouse igg h l, by Thermo Fisher Scientific (2 mentions)
Antifade mounting medium, by Beyotime (2 mentions)
Cell light edu apollo 488 in vitro imaging kit, by RiboBio (1 mentions)
A8020, by Solarbio (1 mentions)
Alexa fluor 488 goat anti rabbit igg h c, by Thermo Fisher Scientific (1 mentions)
5 protocols using sp5ii clsm microscope
1
EdU Incorporation and Hoechst Staining
2
Visualizing NQO1 Localization in Breast Cancer
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IF staining was used to detect the sub-cellular localization of NQO1 protein in breast cancer cells. All steps were performed at RT. Breast cancer cells were grown on coverslips to 70–80% confluence, then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100 for 10 min after 24 h. After blocking with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the E-cadherin, Vimentin antibody at 4 °C overnight, and followed by incubation with Alexa Fluor® 568 goat anti-mouse IgG (H + L) (A11004, 1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h. After washing with PBS, cells were counterstained with 49–6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime)27 (link). Finally, the IF signals were visualized under a Leica SP5II CLSM microscope (Heidelberg, Germany) with filters for the corresponding fluorescent stains.
3
Immunofluorescence Staining of NQO1 in MCF-7 Cells
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IF staining was used to detect the sub–cellular localization of NQO1 protein in MCF-7 breast cancer cells. All steps were performed at RT. MCF-7 cells were grown on coverslips to 70–80% confluence, then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100 for 10 min after 24 h. After blocking with 3% Albumin Bovine V (A8020, Solarbio, Beijing, China) for 1 h, the slides were quickly and gently washed with PBS. The cells were then incubated with the NQO1 antibody (1:500) at 4°C overnight, and followed by incubation with Alexa Fluor® 568 goat anti-mouse IgG (H + L) (A11004, 1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h. After washing with PBS, cells were counterstained with 49-6-diamidino-2-phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and the coverslips were mounted with Antifade Mounting Medium (P0126, Beyotime)
[18 (link)]. Finally, the IF signals were visualized under a Leica SP5II CLSM microscope (Heidelberg, Germany) with filters for the corresponding fluorescent stains.
[18 (link)]. Finally, the IF signals were visualized under a Leica SP5II CLSM microscope (Heidelberg, Germany) with filters for the corresponding fluorescent stains.
4
Subcellular Localization of Paip1 Protein
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IF staining was used to analyze the subcellular localization of the Paip1 protein in MGC-803 and SGC-7901 cells. The cells were grown on coverslips to 65–75% confluence, fixed with 3.65% paraformaldehyde for 5 min and penetrated with 0.5% TritonX-100 for 12 min. After blocking with 3% bovine serum albumin fraction V (A8020, Solarbio, Beijing, China) for 1.5 h, the slides were quickly washed with phosphate-buffered saline (PBS) three times. Then, the cells were incubated overnight at 4 °C with a primary antibody followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (H + C) (A11008, 1:1000 dilution, Invitrogen, USA) for 1 h. After washing with PBS, the cells were counterstained with diamidino phenylindole (DAPI) (C1006, Beyotime, Shanghai, China) and fixed on a slide. Finally, the IF signals were observed and imaged using a Leica SP5II CLSM microscope (Heidelberg, Germany) with filters for the corresponding fluorescent stains.
5
Measuring Cell Proliferation by EdU Assay
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MGC-803 and SGC-7901 cells transfected with Paip1 or siCON were plated in 96-well plates at a density of 5×104 cells/well and then treated with 50 mM EdU for an additional 2 h at room temperature. Then, the cells were fixed with 4% paraformaldehyde for 15 min. After washing with 3% BSA (bovine serum albumin), the cells were permeabilized with 0.5% Triton X-100 for 20 min. After washing with PBS, 500 ml Click-iT mix was added to each well, and the cells were incubated for 30 min. Finally, the cells were stained with 1 ml Hoechst 33342 for 15 min and observed under a Leica SP5II CLSM microscope.
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