After cells were grown on glass coverslips and treated with 10 mmol/L metformin, cells were then fixed with 4% paraformaldehyde diluted in PBS at indicated time points. For immunostaining, cells were blocked with PBS containing 5% normal goat serum and 0.3% Triton X‐100 for 60 min. They were then incubated with antibodies against acetyl‐p53 (K382) (Abcam, Cambridge, UK) diluted in PBS containing 1% BSA and 0.3% Triton X‐100 at 4°C overnight. After washing three times with PBS, slides were incubated with secondary antibodies (AlexaFluor488 goat anti‐rabbit, Invitrogen) at room temperature for 1 hour. Slides were then washed with PBS, counterstained with ProLong® gold antifade reagent (No. P36930, Invitrogen) with 4′,6‐diamidino‐2‐phenylindole (DAPI, Cell Signaling Technology) and analysed by confocal microscopy.
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Japan, Germany, Canada
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is a fluorescent dye used to stain cell nuclei. It binds strongly to DNA and emits blue fluorescence when excited by ultraviolet light. DAPI is a commonly used reagent in fluorescence microscopy and flow cytometry applications.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (10 mentions)
Lsm 880, by Zeiss (10 mentions)
Triton x 100, by Merck Group (8 mentions)
Lsm 700 confocal microscope, by Zeiss (8 mentions)
Prolong gold antifade reagent, by Cell Signaling Technology (6 mentions)
Fluorescence microscope, by Olympus (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Lsm 510, by Zeiss (6 mentions)
Lsm 710, by Zeiss (6 mentions)
Triton x 100, by Solarbio (5 mentions)
Leica tcs sp8 laser confocal microscope, by Leica camera (5 mentions)
Fluorescence mounting medium, by Agilent Technologies (4 mentions)
Bovine serum albumin (bsa), by Solarbio (4 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa, by BD (4 mentions)
40 μm cell strainer, by Corning (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Thermobrite, by Abbott (3 mentions)
254 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Metformin Induces Acetylation of p53
2
Quantifying DNA Damage Response Using γH2AX
3
Immunofluorescence Analysis of Osteoblast Differentiation
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Cells were seeded into six-well plates containing glass cover slides at a density of 2.08 × 104 cells/cm2 and grown to 95 % confluence. The cells received osteoblast differentiation medium containing TEN for 2 weeks. Following fixation in 4 % paraformaldehyde at room temperature for 30 min, the cells were washed three times with PBST (0.1 % Triton X-100 in PBS) and blocked in PBS-bovine serum albumin (1 % bovine serum albumin in PBS) for 1 h at room temperature. Cells were incubated with antibodies against collagen Iα1 (Col Iα1) (Abcam, Cambridge, MA, USA) for 1 h at room temperature. The samples were then washed three times in PBS for 10 min each and incubated with anti-rabbit immunoglobulin G (H+L), F(ab’) 2 Fragment Alexa Fluor 594 conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. For nuclear staining, cells were counterstained with 4’,6-diamidino-2 phenylindole (DAPI, Cell Signaling Technology) for 5 min. Cells were examined using a laser confocal microscope (FV1000; Olympus Optical, Tokyo, Japan). Positive cells were evaluated using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) to measure cellular fluorescence intensity. Cells with a fluorescence intensity ≥150 % of background were considered positive.
4
Immunofluorescent Detection of Collagen I
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MC3T3-E1 cells were seeded into six-well plates containing glass cover slides at a density of 2×105 cells/well and grown to 95% confluence. The cells received osteoblast differentiation medium containing BN for 2 weeks. Following fixation in 4% paraformaldehyde at room temperature for 30 minutes, the cells were washed three times with PBST (0.1% Triton X-100 in PBS) and blocked in PBS-bovine serum albumin (1% bovine serum albumin in PBS) for 1 hour at room temperature. Cells were incubated with antibodies against collagen Iα1 (product code ab21286, diluted 1:100, Abcam, Cambridge, MA, USA) for 1 hour at room temperature. The samples were then washed three times in PBS for 10 minutes each, and incubated with anti-rabbit immunoglobulin G (H+L), F (ab′)2 Fragment Alexa Fluor 594 conjugated secondary antibodies (product code 8889, diluted 1:100, Cell Signaling Technology) for 1 hour at room temperature. For nuclear staining, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology) for 5 minutes. Cells were examined using a laser confocal microscope (FV1000, Olympus Optical Co Ltd, Tokyo, Japan). Positive cells were evaluated using Image-Pro Plus software (Media Cybernetics Inc, Rockville, MD, USA) to measure cellular fluorescence intensity. Cells with fluorescence intensity ≥150% of background were considered positive.
5
Cell Adhesion and Morphology on Hydrogel Substrates
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The SKOV-3 cells were seeded on small circular glass sheets, coated with hydrogel substrates of different stiffness (Matrigen, United States) and were cultured for 48 h. The cells were washed with PBS three times and then were fixed using 4% paraformaldehyde solution for 30 min. After washing with PBS three times, the cells were treated with the 2.5% Triton-XTM for 30 min. The cells were blocked with 3% bovine serum albumin (BSA) (Solarbio, China) and were incubated with a 1:100 dilution of primary antibody against α-tubulin (DM1A-3873s, Cell Signaling Technology, United States), paxillin (sc-365379, Santa Cruz, United States), vimentin (sc-6260, Santa Cruz), E-cadherin (sc-21791, Santa Cruz), β-catenin (sc-7963, Santa Cruz), YAP (sc-101199, Santa Cruz) overnight at 4°C. The cells were then washed with PBS three times and incubated with a secondary antibody (ab150113, Abcam, United Kingdom) for 1 h. 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology), which was diluted with PBS to 1 μg/mL, was added and incubate for 5 min, then washed with PBS three times. Finally, images were acquired using a laser scanning confocal microscope (UltraVIEW VoX; PerkinElmer, United States).
6
Stilbenedisulfonate Inhibitors of NF-κB
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Inhibitor compounds were dissolved in DMSO and used at the indicated concentrations. The N-cyanosulfonamide S0859 (Cayman) and stilbenedisulfonates 4,4’-diisothiocyanatostilbene-2,2’-disulfonate (DIDS, Sigma), 4,4’-diaminostilbene-2,2’-disulfonate (DADS, Sigma), and 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonate (SITS, Sigma) were commercially available, while the DIDS analog 4,4’-ocatanamidostilbene-2,2’-disulfonate (OADS) was synthesized by the Ashfeld group as previously described (Howery et al., 2012 (link)). Antibodies to NF-κB (Rb α NF-κB p65, Cell Signaling Technology D14E12, RRID:AB_10859369), rabbit IgG (Gt α Rb IgG AlexaFluor 488, Invitrogen, RRID:AB_143165), and SLC4A7 (Rb α SLC4A7, Fisher PA557433, RRID:AB_2647530) were used at the indicated concentrations. The nuclear stain 4′,6-diamidino-2-phenylindole (DAPI, Cell Signaling Technology) and actin stain rhodamine-phalloidin (Thermo-Fisher) were used at the indicated concentrations.
7
Quantification of Microglial Activation Phenotypes
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After preparing paraffin sections, the slides were incubated with a series of primary antibodies, including Iba1, CD86 (Zen-bio, China), and CD206 (Proteintech, China). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Cell Signaling, USA). Finally, the sections were imaged with a laser confocal microscope (IXplore SPinSR10, Olympus, Japan) and quantified with the ImageJ software. ImageJ circled the region of interest (ROI), counted the co-labeled cells within ROI area, then determined the percentage of co-labeled cells. The result was expressed as a percentage: co-labeled cells (100%) = (total number of double positive cells/total number of Iba1+ cells × 100%).16 (link)
8
Localization of circAFF1 in LECs
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LECs were seeded in a 4-chanmber slide for 24 h, fixed with 4% paraformaldehyde (PFA), permeabilized with TritonX-100 and incubated with RNA probes at 42 °C in the dark overnight. The slides were washed with 2× saline sodium citrate (SSC) buffer, then the nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, Cell Signaling Technology, Boston, MA, USA). An EVOSTM Microscope M5000 Imaging System (Invitrogen) was used for visualization. The U6 and 18S ribosome probes were used as nuclei and cytoplasm controls, respectively. The circAFF1 probe and control probe were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China).
9
Immunofluorescence Analysis of HUVECs
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Immunofluorescence analysis was performed as described previously [46 (link)]. Briefly, HUVECs at a density of 1 × 104 cells/well were cultured in an eight-well glass Nunc Lab-Tek chamber (ThermoFisher Scientific, Waltham, MA), and treated with HG (30 mM) in the presence or absence of ADC (10 µM) or RES (5 µM) for 1 or 72 h. After treatment, culture medium was removed and cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, washed and blocked with 10% FBS in PBS, and then incubated overnight with the corresponding primary antibodies (1:200) in 1.5% FBS. The cells were then incubated with the fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:1000) (Alexa fluor 488, ThermoFisher Scientific) for another 1 h in 6% bovine serum albumin (BSA). Then, the cells were stained with 1 μg/mL 4’,6-diamidino-2-phenylindole (DAPI, Cell Signaling Technology) for 5 min, washed with PBS, and visualized using a fluorescence microscope (Motic Electric Group) at 40 × magnification.
10
Immunohistochemical Staining of Neural Markers
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Primary antibodies included mouse anti-glutamine synthetase (GS) (MAB302, 1:1000; Millipore, Darmstadt, Germany), mouse anti-glial fibrillary acidic protein (GFAP) (3670, 1:200; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-GFAP (Z0334, 1:500; Dako, Carpinteria, CA, USA), rabbit anti-IBA-1 (019-19741, 1:500; Wako, Richmond, VA, USA), rabbit anti-VEGF (A20, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-PAX6 (1:500; developed by Kawakami, Developmental Studies Hybridoma Iowa City, IA, USA), and rabbit anti-Vimentin (ab45939, 1:300; ABCAM, Cambridge, MA, USA). Secondary antibodies were used at 1:500 room temperature (RT) or 1:1000 4°C overnight (Jackson ImmunoResearch, West Grove, PA, USA). Stains included Isolectin GS-B4 Alexa 568 (I21412, 1:200; Invitrogen, Grand Island, NY, USA), 4′,6-diamidino-2-phenylindole (DAPI) (4083, 1:50,000 from 1 mg/mL stock; Cell Signaling Technology), DRAQ5 (62251, 1:2000; Thermo Scientific, Waltham, MA, USA).
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