Cells were washed with PBS twice for 5 minutes each and fixed with 4% paraformaldehyde for 10 minutes. Subsequently, cells were washed with PBS three times for 5 minutes each. Then, 10% normal goat serum (Gibco Cell Culture, Carlsbad, CA, USA) was added for blocking for 1 hour at room temperature. The primary anti-GSDMD-NT antibody (Amyjet Scientific, Inc., Wuhan, China) was added to the cells, and the cells were then incubated at 4°C overnight. Subsequently, a fluorescein isothiocyanate (FITC)-labeled secondary antibody (Thermo Fisher Scientific, Inc., Rockford, IL, USA) was incubated with the cells for 30 minutes at room temperature. After washing the cells in PBS three times for 5 minutes each, 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei at room temperature for 15 minutes. The residual DAPI was removed using three 5-minute washes with PBS. Images were immediately captured with a fluorescence microscope (Olympus, Tokyo, Japan).
Fluorescein isothiocyanate fitc labeled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescein isothiocyanate (FITC)-labeled secondary antibody is a commonly used fluorescent labeling reagent. It is designed to bind to the Fc region of primary antibodies, allowing for the detection and visualization of target molecules in various immunological techniques, such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (1 mentions)
Pierce quantitative peroxide assay kit, by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay reagent, by Promega (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Tumor necrosis factor alpha tnf α, by R&D Systems (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride edc hcl, by Merck Group (1 mentions)
4 4 6 dimethoxy 1 3 5 triazin 2 yl 4 methylmorpholinium chloride, by Tokyo Chemical Industry (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Bovine type 2 collagen, by Chondrex (1 mentions)
Alamarblue cell viability assay reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using fluorescein isothiocyanate fitc labeled secondary antibody
1
Immunofluorescence Analysis of GSDMD-NT
2
Quantifying Hippocampal Neurogenesis via DCX Immunofluorescence
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In the present study, the level of hippocampal neurogenesis in mice was determined by doublecortin (DCX) immunofluorescence, as mentioned in two previous studies (Jiang et al., 2019 (link); Guan et al., 2021 (link)). In brief, mice anesthetized with 0.5% sodium pentobarbital were subjected to transcardial perfusion of 4%paraformaldehyde. After post-fixation and dehydration, 25 μm of hippocampal slices were collected. For DCX staining, the slices were dealt in a commonly adopted manner: 1, 0.3% Triton X-100 incubation; 2, 3% BSA incubation; 3, primary antibody incubation; 4, washed in PBS; 5, secondary antibody incubation; 6, washed in PBS; 7, 4’,6-diamidino-2-phenylindole (DAPI) incubation; 8, washed in PBS; 9, coverslipped and observed. Primary antibody against DCX (1:100; Cell signaling) and fluorescein isothiocyanate (FITC)-labeled secondary antibody (1:50; Thermo Fisher, Waltham, United States) were used. The method of DCX examination has been described as described before (Jiang et al., 2019 (link)).
3
HA-based Biomaterial Characterization
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HA (90 kDa) was kindly donated by JNC Corporation (Japan). EGCG was obtained from DSM Nutritional Products (Switzerland). Dextran (100 kDa), bovine serum albumin (BSA), phosphate-buffered saline (PBS) and incomplete Freund's adjuvant were purchased from Sigma-Aldrich (USA). Fetal bovine serum (FBS) and PicoGreen dsDNA assay kit were purchased from Thermo Fisher (Singapore). Tumor necrosis factor-alpha (TNFα) was purchased from R&D System (USA). Bovine type II collagen was obtained from Chondrex (USA). 2-(N-Morpholino)ethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 5-aminofluorescein (AF) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (Singapore). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was acquired from Tokyo Chemical Industry Co. Ltd. (Japan). Cyanine7.5 amine dye (Cy7.5) was obtained from Lumiprobe Corporation (USA). Rat anti-human CD44 antibody (clone Hermes-1), isotype control antibody, fluorescein isothiocyanate (FITC)-labeled secondary antibody, Pierce™ quantitative peroxide assay kit and alamarBlue® cell viability assay reagent were purchased from Thermo Fisher Scientific (Singapore). CellTiter-Glo® luminescent cell viability assay reagent was obtained from Promega (Singapore).
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