H9c2 rat cardiomyocyte cell line was get from Wuhan Punosi Life Science and Technology Co., Ltd (Wuhan, China). DMEM low-sugar medium and FBS was purchased from HyClone (Logan, UT). The CCK-8 was purchased from Dojindo (Kumamoto, Japan). LDH, CK-MB, ROS, and GSH test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Iron assay kit and GPX4 primary antibodies were purchased from Abcam (Cambridge, USA). Rabbit anti-rat primary antibodies DNMT-1 (catalog number 24206–1-AP), GPX4 (catalog number 67763-1-Ig), P62 (catalog number 18420-1-AP), Beclin-1 (catalog number 11306-1-AP), GAPDH (catalog number 60004-1-Ig), and Cy3- (catalog number SA00009-2) and FITC- (catalog number SA00003-2)-labeled goat anti-rabbit secondary antibodies were obtained from ProteinTech (Wuhan, China). Rabbit anti-rat primary antibodies NCOA4 (catalog number A5695) and FTH (catalog number A19544) were obtained from ABclonal (Wuhan, China). Second antibodies were purchased from LI-COR Biosciences (IRDye 800CW; catalog number 926-32219, LI-COR Corporate, USA). DNMT-1 inhibitor 5-aza-CdR were purchased from Selleck (Houston, USA). Lipofectamine 2000 was obtained from Invitrogen (San Diego, USA).
Fluorescein isothiocyanate (fitc)
Manufactured by Proteintech
Sourced in United States
FITC (Fluorescein Isothiocyanate) is a fluorescent dye commonly used as a labeling agent in various applications, such as immunohistochemistry, flow cytometry, and fluorescence microscopy. It is a small, reactive molecule that can be covalently attached to proteins, antibodies, or other biomolecules, allowing for their detection and visualization under appropriate excitation and emission wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Ncoa4, by ABclonal (1 mentions)
Iron assay kit, by Abcam (1 mentions)
Dnmt1, by Proteintech (1 mentions)
Cyanine3 (cy3), by Proteintech (1 mentions)
5 aza cdr, by Selleck Chemicals (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Gapdh, by Proteintech (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Ck mb, by Nanjing Jiancheng (1 mentions)
Ab18203, by Abcam (1 mentions)
Cy3 conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Dm4000b, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Rabbit anti mouse cd11b antibody, by Abcam (1 mentions)
3 protocols using fluorescein isothiocyanate (fitc)
1
Cardioprotective Effects of DNMT-1 Inhibition
2
Immunofluorescence Analysis of N-cadherin and NF-κB
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Cells (5 × 104) were plated on coverslips to culture for 12 h. Then the cells were fixed with 2.0% formaldehyde for 30 min, washed three times with PBS, and treated with PBS containing 0.5% Triton X-100 for 20 min. Next, cells were stained with primary antibodies against N-cadherin (ab18203; Abcam) or NF‐κB p65 (#8242; CST) overnight at 4°C. Following washing three times with PBS, the cells were incubated with FITC or Cy3-conjugated goat anti-rabbit IgG (1:200; Proteintech Group) for 1 h and then counterstained with 4’,6-di-amidino-2-phenylindole (DAPI; Sigma Chemicals, USA). Plates were taken using a fluorescence microscope (DM4000B; Leica, Germany) and analyzed by ImageJ software (v1.49).
3
Immunofluorescence Staining of Microglia
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The primary microglial and BV-2 cells were seeded on coverslips and cultured in DMEM with 10% FBS for 24 h. After LPS treatment, cells were washed twice in PBS and xed in 4% paraformaldehyde at room temperature for 30 min, then permeabilized with Triton X-100 for 10 min and blocked with normal goat serum for 1 h. The cells were stained by rabbit anti-mouse CD11b antibody (1:200; Abcam, USA) overnight at 4°C and then incubated with uroscence-secondary antibody FITC (1:500; Proteintech, USA). Cells were treated with DAPI solution for 20 min at 37°C. Images were acquired by a Fluorescence confocal microscope (Lecia SP8, Germany ) and analyzed with ImageJ software system.
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