Cells were washed twice with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 48-h after transfection. After blocking with 5% BSA for 30 min, the cells were incubated with an anti-E-cadherin (Proteintech, diluted 1:100) antibody or an anti-vimentin (Proteintech, diluted 1:100) antibody at 4 °C overnight. The secondary antibody, Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen, diluted 1:1000), was then added. DAPI (Meilunbio, China) was used to visualize nuclei. The cells were observed using a confocal microscope (Olympus, Japan).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Meilun
Sourced in China
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye used for staining and visualizing nucleic acids, specifically DNA, in biological samples. It is a widely used tool in various applications such as cell biology, molecular biology, and microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (3 mentions)
Anti vimentin, by Proteintech (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Fitc or cy3 labeled secondary antibodies, by Proteintech (1 mentions)
Image pro plus software, by Olympus (1 mentions)
A confocal fluorescence microscope, by Zeiss (1 mentions)
Phalloidin solution, by Meilun (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Bioss Antibodies (1 mentions)
Ts100 f, by Nikon (1 mentions)
Gsdmd, by Affinity Biosciences (1 mentions)
Leica tcs sp8 confocal laser scanning microscope, by Leica Microsystems (1 mentions)
Anti cd31, by Proteintech (1 mentions)
Fitc phalloidin, by Solarbio (1 mentions)
Anti α sma, by Proteintech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Glutaraldehyde, by Solarbio (1 mentions)
Ripa lysis buffer, by Meilun (1 mentions)
23 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunofluorescence Analysis of E-cadherin and Vimentin
2
Immunofluorescence Staining of M1 and M2 Macrophages
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Immunofluorescence staining was conducted as previously described [33 (link)] to detect M1 macrophage (CD86) and M2 macrophage (CD206) infiltration in skeletal muscle. Skeletal muscle sections were subjected to antigen retrieval, and the sections were blocked and labeled overnight at 4 °C with rabbit anti-CD86 (1:200, Cat. 13395-1-AP, Proteintech) and mouse anti-CD206 (1:200, Cat. sc-58986, Santa Cruz). After the sections were washed in PBS three times, FITC- or Cy3-labeled secondary antibodies (1:200, Proteintech) were used for the final immunostaining. Sections that were not incubated with primary antibodies were used as negative controls. The sections were incubated with DAPI (Meilunbio) to stain nuclei and examined under a fluorescence microscope (Olympus).
3
Quantifying Apoptosis in Skeletal Muscle
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Apoptotic nuclei in skeletal muscle were examined using double-fluorescent labeling of TUNEL and dystrophin. TUNEL staining was performed according to the manufacturer’s protocol (Roche Inc., Basel, Switzerland). After TUNEL labeling, tissue sections were incubated with a rabbit anti-dystrophin monoclonal antibody (1:200, Cat. 12715-1-AP, Proteintech) followed by an anti-rabbit IgG cyanin 3 (Cy3) (1:200, Cat. SA00009-2, Proteintech). Sections were incubated with DAPI (Meilunbio, Dalian, China) to stain nuclei and examined under a fluorescence microscope (Olympus, Tokyo, Japan). Photomicrographs were merged and saved by Image-Pro Plus software (Olympus). The numbers of TUNEL- and DAPI-positive nuclei were counted and only labeled nuclei that colocalized with dystrophin staining were counted. The data are expressed as the TUNEL index, which was calculated by counting the number of TUNEL-positive nuclei divided by the total number of nuclei. The TUNEL index for each muscle was calculated from five random, nonoverlapping fields at an objective magnification of 40×.
4
Visualizing Vascular Smooth Muscle Cytoskeleton
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SMCs were stimulated with 10 ng/mL PDGF-BB for 24 h, seeded onto nanofiber scaffold mats at a density of 2 × 104/well, and cultured for 48 h. The cultured cells were fixed with 4% PFA overnight and stained with phalloidin solution for 60 min according to manufacturer instructions (Meilun Bio, Dalian, China). Cells were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Bioss, Shanghai, China) for 10 min. Similarly, SMCs were seeded onto 24-well plates at a density of 1.5 × 104/well and cultured for 24 h. The cultured SMCs were then starved for 24 h, and the cells were fixed and stained with phalloidin solution (Meilun Bio) and DAPI, as previously described. A confocal fluorescence microscope (Carl Zeiss) was used to observe the cytoskeleton.
5
Measuring Active β-Catenin in TFK1 and RBE Cells
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TFK1 and RBE cells (1×105 cells/well) were incubated in the 12-well plate, and the concentrations of NBT (50 μmoL/L) combined with cells were cultured for 24 hours, then fixed with 4% paraformaldehyde. After 0.1% Triton X-100 breaks the cell membrane, samples are blocked with 10% BSA. Active β-catenin diluted in 1% BSA was added and incubated overnight. After repeated washes with PBS, the samples were incubated with a rabbit-anti-human antibody diluted in 1% BSA (1:200). Finally, the cells were stained with DAPI (Meilunbio, MA0222, China) dyeing solution, then the cells were imaged by the inverted fluorescence microscope (Nikon, TS100-F, Japan).
6
Immunofluorescence Analysis of Ovarian Tissue
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The ovarian tissues were fixed in 4% paraformaldehyde solution, then dehydrated and embedded in paraffin. The tissues were sectioned at 5 µm. The slides were dewaxed in xylene and rehydrated through graded ethanol concentrations. Antigen retrieval was performed in citrate solution and then were blocked with 3% BSA for 1 h. After incubating with the primary antibody (CD3, Affinity Biosciences, DF6594; CD14, ABclonal, A19011; DDX4, Abcam, ab27591, GSDMD, Affinity, AF4012) overnight at 4 °C, the slides were incubated with appropriate secondary antibody (CY3 goat anti‐mouse IgG, EarthOx, E031610‐01; CY3 goat anti‐rabbit IgG,EarthOx, E031620‐01; DyLight 488 goat anti‐rabbit IgG, EarthOx, E032220‐01; Alexa Fluor 647 goat anti‐mouse, Abcam, ab150115) for 1 h at room temperature, followed by staining with 4′,6‐diamidino‐2‐phenylindole (DAPI, meilunbio) and imaged with Leica TCS‐SP8 Confocal Laser Scanning Microscope (Leica Microsystems).
7
Biomimetic Scaffold Fabrication and Characterization
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ALA (≥ 92.5%) was friendly provided by Davisco Food International, USA. LZM (20,000 U/mg), BSA (≥ 98%) and COL (> 90%) were purchased from MeilunBio (Dalian, China). PCL (Mn = 80,000, Sigma Aldrich, MO, USA), hexafluoroisopropanol (HFIP, Rhawn, Shanghai, China). FITC-phalloidin, DAPI and glutaraldehyde were purchased from Solarbio (Beijing, China). α-MEM culture medium, DAPI and RIPA lysis buffer were purchased from Meilunbio. Anti-CD31 and anti-α-SMA were obtained from Proteintech (IL, USA). Fetal bovine serum (FBS) was obtained from Gibco (NY, USA). Cy3-conjugated anti-rat IgG and FITC-conjugated anti-rat IgG were purchased from Servicebio (Wuhan, China). Collagen spongeⓇ was obtained from BIOT Biology (Wuxi, China).
8
Matrigel-based cell adhesion assay
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In a 96-well plate with Matrigel (BD, USA) already spread. 2000 cells were seeded in each well, and serum-free medium was used to cultivate the cells. After 2 h of incubation, cells adhered to Matrigel were washed twice with PBS. After fixation with 4% paraformaldehyde (Meilunbio, China) for 10 min, DAPI (Meilunbio, China) staining was used to observe the number of adherent cells under a fluorescence microscope (Olympus, Japan). Each experiment was repeated at least at least 3 times.
9
Macrophage Migration Assay with Exosomes
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Macrophages were cultured in medium with 10% exosome-depleted FBS on 12-well plates and scratch wounds were created using a 200 μl pipette tip per well before the PBS/exo/exo-T was added. The initial wound size of macrophages was measured immediately after washing the cells twice. After 24 h, images were captured again in the same fields. The area of migration was calculated as the percentage wound closure using ImageJ. The transwell assay was performed in 8 μm pore-size inserts (Corning Incorporated, Corning, CA, USA). THP-1 cells differentiated into macrophages and were cultured in the apical chamber. After 24 h of coculture with PBS/exo/exo-T in the incubator, the migratory macrophages at the bottom of the chamber were stained with DAPI (Dalian Meilun Biotechnology, Dalian, China) after fixation and were visualized using a High Content Imaging System.
10
Evaluating Nanoxel-PM Delivery Efficacy
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DTX, ginsenoside 20 (S)–Rg3, and Nanoxel-PM (Samyang Biopharm) were gifted by Bensu Medicine Technology Co. Ltd. (Shanghai, China). Cholesterol was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). PL-100M was purchased from AVT Medicine Technology Co. Ltd. (Shanghai, China). Coumarin-6 (C6), Hoechst 3334w, DAPI, 1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), DiD, MTT, and 1% crystal violet solution were purchased from Meilun Biotechnology Co., China. Annexin V–FITC/propidium iodide (PI) apoptosis detection kit and phosphatase inhibitor cocktail were provided by Yeasen Biotechnology Co., China. Penicillin, streptomycin, and 0.25% trypsin-EDTA were purchased from Invitrogen Co., USA. Protein GLUT1 was obtained from Wuhao Bio-Tech Co., China. CM5 sensor chips were purchased from GE Healthcare Bio-Sciences AB (Sweden). Anti-Glut1 small interfering RNA (siRNA) (CCAACUGGACCUCAAACUUTT, AAGUUUGAGGUCCAGU-UGGTT) and siRNA-mate were provided by GenePharma Co., China. All other chemicals and solvents used in this study were of reagent grade or high-performance liquid chromatography (HPLC) grade.
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