NIH/3T3 cells were seeded on coverslips. Cells were fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich) at room temperature for 15 min. Fixed cells were washed with PBS three times. Then, cells were incubated overnight with primary antibodies in permeabilizing solution (1% bovine serum albumin, and 0.2% triton X-100 in PBS) at 4°C. After washing cells, fluorescence-conjugated secondary antibodies were added and cells were incubated at room temperature for 1 h. Nuclei were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich). The coverslips were mounted on slide glasses using fluorescent mounting medium (S3023, Dako, CA, USA). Antibodies used for immunocytochemistry are given in Supplementary Table 3 . Images were obtained using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany) equipped with 40x/1.2 (water-immersed) and 63x/1.4 (oil-immersed) objective lenses. Image acquisition was performed using the ZEN software (Carl Zeiss). To calculate the number of ciliated cells, nine non-overlapping fields were selected randomly, and at least 100 cells were counted. Brightness and contrast of images were adjusted using Photoshop CC 2015 (Adobe, CA, USA).
Dapi d9542
Manufactured by Merck Group
Sourced in United States, Germany
DAPI (D9542) is a fluorescent stain used for labeling and visualizing DNA in biological samples. It is commonly used in fluorescence microscopy and flow cytometry applications to detect and quantify cellular DNA content.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Fv3000, by Olympus (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ab178566, by Abcam (2 mentions)
Inverted fluorescence microscope, by Olympus (2 mentions)
Anti β actin a5441, by Merck Group (2 mentions)
Matrigel 354234, by BD (2 mentions)
Biocoat cell inserts, by BD (2 mentions)
Cell lysis buffer 9803, by Cell Signaling Technology (2 mentions)
Ab15580, by Abcam (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
61 protocols using dapi d9542
1
Immunocytochemistry of Ciliated NIH/3T3 Cells
2
Immunofluorescence Analysis of DBC1 and SUMO1
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Cells were seeded on 24-well glass slides. After PBS wash, cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.3% TritonX-100 for 15 minutes, and blocked with normal donkey serum for 1 hour. Then the slides were incubated with the anti-DBC1 (5857S, Cell Signaling Technology.) and anti-SUMO1 antibody (S8070, Sigma) or normal mouse IgG at 4°C overnight. After the PBS washings, the slides were incubated with secondary antibody (4412S, 8890S, Cell Signaling Technology). Cell nuclei were stained with DAPI (D9542, SIGMA) for 5 min. Slides were mounted with anti-fade fluorescent mounting medium (Southern Biotech). Images were captured with a TissueFAXS Q confocal microscope (TissueGnostics, Vienna, Austria). Images were analyzed by TissueFAX Viewer software.
3
Immunostaining of Mouse Brain Sections
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Mouse brains were fixed in 4% PFA in phosphate-buffered saline (PBS) overnight, incubated in 25–30% sucrose in PBS, embedded in OCT (SAKURA, USA), and stored at –80 °C until use. Brains were sectioned (14–16 μm) using a cryostat. For antigen recovery, sections were incubated in heated (95–100 °C) antigen recovery solution (1 mM EDTA, 5 mM Tris, pH 8.0) for 15–20 min, and cooled down for 20–30 min. Before applying antibodies, sections were blocked in 10% normal goat serum in PBS with 0.1% Tween-20 (PBT) for 1 h. Sections were incubated with primary antibodies at 4 °C overnight and visualized using goat anti-rabbit IgG–Alexa-Fluor-488 (Jackson ImmunoResearch, UK) and/or goat anti-mouse IgG–Alexa-Fluor-546 (Jackson ImmunoResearch, UK) (1:300, Molecular Probes) for 1.5 h at room temperature. Images were captured using a Leica digital camera under a fluorescent microscope (Leica DMI6000B).
Primary antibodies against the following antigens were used: BrdU (1:100) (Abcam, UK), Ki67 (1:300) (Abcam, UK), Sox2 (1:100) (Santa Cruz Biotechnology, USA), Pax6 (PRB-278P, 1:200, BioLegend), Tbr1 (1:2,000) (Abcam, UK), Tbr2 (1:500) (Abcam, UK), NeuN (1:3,000) (Abcam, UK), Satb2 (1:500) (Abcam, UK), Caspase3 (1:100, Abcam), and DAPI (D9542, 1:1000, Sigma), also see Supplementary Table S14 .
Primary antibodies against the following antigens were used: BrdU (1:100) (Abcam, UK), Ki67 (1:300) (Abcam, UK), Sox2 (1:100) (Santa Cruz Biotechnology, USA), Pax6 (PRB-278P, 1:200, BioLegend), Tbr1 (1:2,000) (Abcam, UK), Tbr2 (1:500) (Abcam, UK), NeuN (1:3,000) (Abcam, UK), Satb2 (1:500) (Abcam, UK), Caspase3 (1:100, Abcam), and DAPI (D9542, 1:1000, Sigma), also see Supplementary Table S
4
Immunofluorescence Staining of H9c2 Cells
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The normal cultured H9c2 cells in the log phase were taken, digested with 0.25% Trypsin, centrifuged at 1,000 rpm for 5 min, counted under a counting plate, spread on a 24-well plate, and 8 × 10 4 cells were added to each well, for a total of six wells. Incubate in an incubator. Cells were grown on a coverslip. The cells were then fixed in 4% paraformaldehyde for 15 min, followed by incubation with permeabilization solution for 30 min at room temperature. The coverslip was washed with PBS three times and incubated with a blocking solution for 1 h at room temperature. Primary antibody in blocking solution was added, cells were incubated overnight at 4°C, washed with blocking solution, and then incubated secondary antibody for 1.5 h at room temperature. The coverslip was washed and mounted with 10 ng/ml DAPI (D9542, sigma, United States) stain and visualized using a fluorescence microscope (IX73, OLYMPUS, Japan).
5
Immunofluorescence Analysis of Cell Cultures
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Cells were plated on sterile glass coverslips for 48 h, fixed with 4% paraformaldehyde (PFA)/PBS for 20 min, and permeabilized in 0.5% Triton X‐100/PBS for 10 min. Cells were blocked for 1 h, incubated overnight (4 °C) with the primary (Table S1 ) and secondary (Table S1 ) antibodies for 1 h at room temperature. DAPI (D9542; Sigma‐Aldrich) staining was performed for 10 min. Confocal images were captured by the inverted microscope Zeiss Axio Observer Z1, equipped with the laser scanning unit LSM 780. Digital images were acquired with zen 2011 software, and statistical analysis was performed by imagej (https://imagej.nih.gov/ij/ ).
6
Oyster Hemocyte Phagocytosis Kinetics
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Oyster hemocytes were cultured in confocal dishes with a suspension at a density of 105 cells/mL and a volume of 500 µL per sample. After 15 min of incubation, cells were infected for 15 min, 30 min, and 60 min by adding pH-sensitive BioParticles (P35361, Thermo Fisher, USA) at a concentration of 1 mg/mL. Cells were then washed three times to remove extracellular particles. After phagocytosis, paraformaldehyde (4%, cold) was used to terminate the phagocytic process and fix cells for 15 min. Cells were washed three times with PBS, followed by DAPI (D9542, Sigma, USA) staining of nuclei for 5 min. For each time point, several pairs of images (generally four) were acquired with appropriate filters by Leica LP8X confocal microscopy. After acquisition, images were analyzed by Image Pro Plus software and corresponding fluorescence intensity data by GraphPad5 software.
7
Murine Macrophage and HSC Assay
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Cells included murine macrophages RAW 264.7 (337875) (BNCC, Beijing, China) and primary murine HSCs (isolated from male SD rats). Reagents included carbon tetrachloride (56235) (Sigma-Aldrich, Shanghai, China), Lipopolysaccharide (LPS; S1732) (Beyotime, Hainan, China), resveratrol (SC0276) (Beyotime, Hainan, China), selisistat (49843-98-3) (EX-527; MedChemExpress, NJ, USA), DAPI (D9542) (Sigma-Aldrich, Shanghai, China), Alexa Fluor™ 647 Phalloidin (A22287) (Thermo, Shanghai, China), protease cocktails inhibitor (P1005) (Beyotime, Hainan, China) and Phenylmethanesulphonyl fluoride (ST506) (PMSF; Beyotime, Hainan, China).
8
Duoxa1 Overexpression in Mouse Cells
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Full-length wild-type Duoxa1 was amplified using PCR from mouse cDNA and ligated into the pMX-IRES-EGFP vector as pMX-Duoxa1 using the BamHI and XhoI (Enzynomics, Daejeon, Korea) sites. The following primers were used: Duoxa1-For, 5′-GCTAGGATCCATGGCTGCTCTTGGACACAC-3′ and Duoxa1-Rev, 5′-CGACTCGAGCAGGGAACAGTCGGACTCTTTG-3′. TRIzol reagent was obtained from Life Technologies (Carlsbad, CA, USA). A monoclonal β-actin (A5441) antibody and DAPI (D9542) were obtained from Sigma (St. Louis, MO, USA). Antibodies for anti-phospho-ERK-1/2, anti-total ERK-1/2, anti-phospho-p38, anti-total p38, anti-phospho-JNK, anti-total JNK, anti-phospho-IκB, anti-phospho-Akt, anti-Akt, anti-phospho-Src, anti-Src, and anti-Btk were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Phospho-Btk (GTX61791) antibody was obtained from GeneTex (Irvine, CA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Duoxa1 antibody was obtained from Bioss Inc (BS-11433®, Wobun, MA, USA). Donkey anti-rabbit and anti-mouse immunoglobulin secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA).
9
Immunofluorescence Analysis of Myofibroblast Markers
10
Immunofluorescent Labeling of Tissue Samples
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Transparent tissue samples were washed in 1 × PBS/0.01% SA for 24 h and then incubated with primary antibodies against tyrosine hydroxylase (1:50, rabbit polyclonal to tyrosine hydroxylase, ab112, Abcam) or platelet endothelial cell adhesion molecule/CD31 (1:10, rabbit polyclonal to CD31, ab28364, Abcam) dissolved in 1 × PBS/0.01% SA for 48 h, followed by washing in 1 × PBS/0.01% SA for 24 h. Samples were then incubated with the secondary antibody (1:100, Alexa 488 anti-rabbit, A11034, Life Technologies or 1:100, Alexa-594 anti-rabbit, A11012, Life Technologies), lectin dye (1:100, Tomato, 594DL-1177, DyLight), and 4′,6-diamidino-2-phenylindole (1:100, DAPI, D9542, Sigma-Aldrich), which were all dissolved in 1 × PBS/0.01% SA, and incubated for 48 h covered by aluminum foil to avoid fluorescence bleaching. Finally, all samples were washed with 1 × PBS/0.01% SA buffer for 24 h.
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