The immunofluorescence method was partially based on our research group’s previously published articles (Wang et al., 2020b (link)). Tumor cells grown on glass slides were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.2% Triton X-100 (Sigma‒Aldrich, Darmstadt, Germany) and washed with PBS containing 0.02% Tween-20 (PBST) three-times. After incubation for 45 min in 10% BSA diluted in PBST, the cells were incubated with CMTM6 antibody (#D260396-0100; Sangon Biotech, Shanghai, China) at 4 °C overnight in a humidified chamber. After washing with PBST three times, the cells were incubated with goat anti-rabbit IgG (diluted 1:150 with 0.01 mol PBS) conjugated with FITC (ZF-0311; Zhong Shan Jin Qiao, Beijing, China) at 37 °C for 40 min in a humidified chamber in the dark. Nuclei were stained with DAPI (C1005; Beyotime, Shanghai, China) at 25 °C for approximately 15–20 min. The images used for analysis were obtained with fluorescence microscopy (Olympus BX51, Tokyo, Japan).
Dapi c1005
Manufactured by Beyotime
Sourced in China
DAPI (C1005) is a fluorescent dye used for the staining of DNA in biological samples. It emits blue fluorescence upon binding to DNA, allowing for the visualization and identification of nuclei in cells.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Leica (3 mentions)
Lsm 700, by Zeiss (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Inverted microscope, by Leica (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Goat serum, by Beyotime (1 mentions)
Mini26 1kt, by Merck Group (1 mentions)
Ripa lysis buffer p0013c, by Beyotime (1 mentions)
High glucose dmem medium 11965092, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Olympus (1 mentions)
P5282, by Merck Group (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
Fv1000, by Olympus (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab275377, by Abcam (1 mentions)
Ab217345, by Abcam (1 mentions)
Ecl kit, by Yeasen (1 mentions)
C1005, by Beyotime (1 mentions)
Tem system, by Hitachi (1 mentions)
26 protocols using dapi c1005
1
Immunofluorescence Staining of Tumor Cells
2
Localization of YAP in Chondrocyte-like iPSCs
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The localization of YAP expression in N-hiPS-Ch was determined via immunofluorescence staining. Briefly, cells were harvested on day 14 and were seeded in 24-well plates at 1 × 104 cells/cm2. After 48 h of treatment with low, medium, and high concentrations of T-2 toxin and DON alone or in combination, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with PBS thrice. The samples were then treated with 0.1% Triton X-100/PBS for 30 min at room temperature and blocked with 1% bovine serum albumin (BSA)/0.3 M glycine at 4 °C for 60 min. Subsequently, the samples were incubated with rabbit anti-YAP antibody (13584-1-AP, 1:100, Proteintech) at 4 °C overnight, followed by secondary fluorescent conjugated secondary antibodies. Cell nuclei were counterstained with DAPI (C1005, Beyotime, Shanghai, China). In addition, the identification of hiPSCs and hiPS-Ch was performed using immunofluorescence staining. The samples were incubated with an anti-Nanog antibody (14295-1-AP, 1:100), anti-OCT4 antibody (ab181557, 1:250), anti-COL2A1 antibody (ab185430, 1:100), and anti-ACAN antibody (13880-1-AP, 1:100). Staining was observed using a fluorescence microscope (Leica Microsystems, Germany) and photographed.
3
Immunofluorescent Staining of SOX2 in GBM Tumorspheres
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GBM#P3 tumorspheres were seeded on poly‐D‐lysine (Sigma‐Aldrich)‐coated coverslips. Cells were fixed in 4% paraformaldehyde in PBS and blocked with 10% goat serum (Beyotime, Shanghai, China) and 0.3% Triton X‐100 in PBS for 30 min. Slides were incubated with mouse monoclonal SOX2 antibody (sc‐365823, 1:100, Santa Cruz Biotechnology; Dallas, TX) overnight and incubated with anti‐mouse IgG‐conjugated Alexa Fluor 568 (A‐11031, 1:500, Invitrogen) for 2 h at room temperature. Nuclei were counterstained with DAPI (C1005, Beyotime; Haimen, Jiangsu, China). Images were captured with a Leica inverted microscope.
4
Visualizing Exosome Uptake by Vascular Smooth Muscle Cells
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To confirm that the AFs-Exos could be taken up by VSMCs, we labeled the exosomes with a red fluorescent dye (PKH26, MINI26-1KT, Sigma), and co-incubated the labeled exosomes with VSMCs as described previously [36 ]. Briefly, 4 µL of PKH26 dye was first dissolved in 500 µL Dilute C solution. 100 µg exosomes were added to the mixture and incubated at room temperature for 5 min. Then 500 µL BSA was added to terminate the reaction. The unbound dyes were removed by ultracentrifugation at 100, 000 g for 70 min. The labeled exosomes were co-incubated with VSMCs at 37℃ for 12 h. After washing with PBS, VSMCs were fixed with 4% paraformaldehyde at room temperature for 30 min. After washing with PBS, VSMCs were incubated with DAPI (C1005, Beyotime Biotechnology, Shanghai, China) for 5 min at room temperature to stain nuclei. After washing with PBS, the red fluorescent signals in VSMCs were detected by the fluorescence microscope.
5
Retinol-Induced Myogenic Differentiation
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ICR mice were purchased from Jilin Changchun YISI Laboratory Animal Technology Company. Mouse C2C12 cells were purchased from Procell (Wuhan, China). Retinol (VitA, R7632, purity ≥ 98%) and Retinoic acid (RA, R2625, purity ≥ 95%) were purchased from Sigma (Kawasaki, Japan). Dimethyl sulfoxide (DMSO, ab146588, purity > 98%) was purchased from abcom (Waltham, MA, USA). VitA powder was dissolved in dimethyl sulfoxide DMSO and diluted to different concentrations to treat C2C12 cell. Fetal bovine serum (FBS002), horse serum (YT2515), penicillin and streptomycin (YT2515) were purchased from Biological Industries (Cromwell, CT, USA). High glucose DMEM medium (11965092) was purchased from Gibco (Canberra, Australia). RIPA lysis buffer (P0013C) and DAPI (C1005) were purchased from Beyotime (Shanghai, China). Hematoxylin-eosin staining kit (G1120) was purchased from Solarbio (Beijing, China). Anti-GAPDH (sc-365062), Anti-MyoG (sc-52903) and Anti-MyoD (sc-377460) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and Anti-RARα (A0370) was purchased from ABclonal (Wuhan, China). Goat anti-Rabbit Anti-IgG (A0208) was purchased from Beyotime (Shanghai, China).
6
Immunofluorescence Imaging of KGN Cells
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KGN cells were cultured with or without 4 μM TubA for 24 h, washed twice with PBS, fixed with 4% paraformaldehyde for 4-8 h at 4 °C, and permeabilized with 0.1% Triton X-100 in PBS for 30-45 min. Subsequently, cells were incubated with the primary antibody for 12-48 h at 4 °C. The primary antibodies were recycled for use no more than three times. Cells were washed twice with PBS at 100 rpm for 15 min. The secondary antibody was then added to the cells and incubated for 1 h at 37 °C followed by 3 washes with PBS for 15 min. Nuclei were stained with DAPI (C1005, Beyotime, China). The cells were again washed 2 times for 5 min and then photographed with the Olympus confocal microscope. Pictures of the control and treatment groups were not altered for intensity and/or contrast. The antibodies used are listed in Supplementary Table 1 .
7
Imaging Cochlear Explants with DAPI and Phalloidin
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The cochlear explants were fixed in 4% paraformaldehyde in 0.01 M PBS for 1 h at room temperature. After washing three times in 0.01 M PBS, explants were stained with DAPI (C1005, Beyotime Institute of Biotechnology, Jiangsu, China) and phalloidin (0.05 mg/mL, P5282, Sigma-Aldrich) for 10 min each. Images were captured with a laser scanning confocal microscope (Nikon, Tokyo, Japan). Three regions from the apical, middle, and basal turns of the stretched cochlear explants were scanned using a ×60 magnification lens.
8
Fluorescent Labeling of PRP-Derived Extracellular Vesicles
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PRP-Exos were labeled with PKH67 dye (Fluorescence Biotechnology, China). Briefly, 100 μg of PRP-Exos were incubated with a staining solution mixture (0.4 μL of probe dissolved in 100 μL dilution) for 5 min at 37 °C, followed by 15 min at 4 °C in the dark. Unbound dye was subsequently removed via centrifugation (100,000×g, 70 min). The TSPCs were incubated with fluorescently labeled PRP-Exos (50 μg/mL) for 24 h. Following fixation with 4% paraformaldehyde fixation (BN20094, Biorigin, China), cells were stained with DAPI (C1005, Beyotime, Beijing, China). The internalization of PRP-Exos by TSPCs was assessed using laser confocal microscopy (FV1000, Olympus, Tokyo, Japan).
9
Immunofluorescence Staining of Gut Tissue
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Paraffin sections of the gut were submerged in xylene and a concentration gradient of ethanol for dewaxing and hydration. After antigen repair, 5% goat serum (16210064, Thermo Fisher Scientific, Massachusetts, USA) was used for blocking. Samples were incubated in FITC solution (1.5 mg/mL) overnight at 4 °C. FITC (HY-66019) was bought from MedChemExpress (New Jersey, USA). After two washes in PBS (pH = 7.4) containing 1% Tween-20 (T104863, Shanghai Aladdin Biochemical Technology, China), the samples were stained with DAPI (C1005, Beyotime Biotechnology, Shanghai, China) in the dark for 3–5 min and glycerin (G8190, Beijing Solarbio Science & Technology, China) was used for sealing.
10
Extracellular Vesicle Characterization and Internalization
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Western blotting: Primary antibodies: anti‐CD63 (ab217345, Abcam, USA), anti‐Alix (ab275377, Abcam), anti‐TSG101 (ab125011, Abcam), anti‐Calnexin (ab313243, Abcam). The membranes were developed using ECL kit (Yeasen, China).
TEM: DsEVs sample was examined by TEM (Hitachi TEM system, Japan).
NTA: 10 µL DsEVs suspension was loaded into the sample chamber of ZetaVIEW S/N 252 (Particle Metrix GmbH, Germany). Data analysis was performed with software (ZetaView 8.04.02, Germany).
Detection of the loading rate of TAT47‐57‐MBP87‐99A91 into DsEVs: After co‐culturing the FITC‐labeled peptide with DCs for 24 h, DCs were collected and stained with phalloidin (Actin‐Tracker Red‐594, C2205S, Beyotime) and 4′,6‐diamidino‐2‐phenylindole (DAPI, C1005, Beyotime, China). After the isolation of DsEVs.
DsEVs internalized by DCs and CD4+ T cells in vitro: DsEVs were labelled with the Dil (Solarbio life sciences, China) and then co‐cultured with DCs or CD4+ T cells for 24 h. Cells were fixed and followed by staining with phalloidin (Actin‐Tracker Green‐488, C2201S, Beyotime) and DAPI (C1005, Beyotime). The images were obtained by scanning confocal fluorescence microscopy.
TEM: DsEVs sample was examined by TEM (Hitachi TEM system, Japan).
NTA: 10 µL DsEVs suspension was loaded into the sample chamber of ZetaVIEW S/N 252 (Particle Metrix GmbH, Germany). Data analysis was performed with software (ZetaView 8.04.02, Germany).
Detection of the loading rate of TAT47‐57‐MBP87‐99A91 into DsEVs: After co‐culturing the FITC‐labeled peptide with DCs for 24 h, DCs were collected and stained with phalloidin (Actin‐Tracker Red‐594, C2205S, Beyotime) and 4′,6‐diamidino‐2‐phenylindole (DAPI, C1005, Beyotime, China). After the isolation of DsEVs.
DsEVs internalized by DCs and CD4+ T cells in vitro: DsEVs were labelled with the Dil (Solarbio life sciences, China) and then co‐cultured with DCs or CD4+ T cells for 24 h. Cells were fixed and followed by staining with phalloidin (Actin‐Tracker Green‐488, C2201S, Beyotime) and DAPI (C1005, Beyotime). The images were obtained by scanning confocal fluorescence microscopy.
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