The primary passage of cells in 12-well chambers was fixed in 4% paraformaldehyde for 30 min at room temperature. After that, Am-DCSCs were permeabilized in 0.3% Triton X-100 for 5 min and blocked in 5% BSA for 1 h. Then, Am-DCSCs were incubated with antibodies against STRO-1 (Novus Biologicals) at 1:150 dilution at 4 °C overnight. Alexa Fluor-648-conjugated anti-IgM (Yeasen) was used as a secondary antibody at 1:200 dilution, and DAPI (Abcam; 1:500) was used for nuclear counterstain. Bm-DCSCs and Am-DCSCs at P3 were stained by Alexa Fluor 555 Phalloidin (Abcam; 1:200) and DAPI (Abcam; 1:500) as well for actin staining. Slides were examined with a confocal laser scanning microscope (CLSM; Leica).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Abcam
Sourced in United Kingdom, United States, China, Japan, Germany
DAPI is a fluorescent dye that binds strongly to DNA. It is commonly used in cell biology and microscopy applications to stain and visualize cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (21 mentions)
Lsm 880, by Zeiss (21 mentions)
Lsm 780 confocal microscope, by Zeiss (13 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Fluorescence microscope, by Olympus (11 mentions)
Lsm 880 confocal microscope, by Zeiss (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Paraformaldehyde (pfa), by Merck Group (8 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Tcs sp8, by Leica camera (7 mentions)
Anti ki67, by Abcam (6 mentions)
Fluorescence microscope, by Zeiss (6 mentions)
Lsm 780, by Zeiss (6 mentions)
Lsm 710, by Zeiss (6 mentions)
Triton x 100, by Solarbio (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Alexa fluor 488, by Abcam (5 mentions)
Ab92547, by Abcam (5 mentions)
Neuronal nuclei (neun), by Abcam (5 mentions)
471 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunofluorescence Staining of Amniotic Stem Cells
2
Immunofluorescence Detection of T-Cell Receptors and IgA-Anti-TG2 Complexes
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Cryosections (4 µm) were incubated overnight at 4 °C with the following combinations of unlabeled mAb: the δ chain of the T-cell receptor (TCR) γ/δ complex (Thermo-Fisher Scientific, clone 5A6. E9, IgG1) combined with mAb to CD3 (clone RIV-9, IgG3), as detailed elsewhere7 (link). A mAb against human transglutaminase-2 (Neomarker/Abcam, clone CUB7402, IgG1) combined with rabbit antiserum against human IgA (Dako, Denmark) was used to detect the colocalization of IgA and TG2, representing IgA-anti-TG2 immune complexes18 (link). The secondary reagents were various combinations of Alexa 488- or 594-conjugated anti-mouse subclass-specific goat antisera in combination with Alexa 488- or Alexa 594-conjugated goat anti-rabbit IgG (all from Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). DAPI (4′,6-diamidino-2-phenylindole; Abcam) was occasionally added as a nuclear fluorescence marker to visualize the intraepithelial location of T cells.
3
Immunofluorescence Imaging of Cx43 and VDAC
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Immunofluorescence staining was performed as described previously.22 (link)
Briefly, MLOY4 cells were cultured in 35 mm confocal dishes at a density of 2 × 105 cells/dish for 24 hours. They were then fixed in 4% paraformaldehyde for 15 minutes, rinsed with PBS three times, permeabilized with 0.5% Triton X-100 (Beyotime) for 15 minutes, and blocked with 5% goat serum for one hour. The samples were then incubated with the anti-Cx43 rabbit monoclonal antibody (1:1,000, Abcam, UK) overnight at 4°C. The secondary antibody was fluorescein isothiocyanate (FITC) fluorescent antibody (1:1,000, Abcam, UK) and TRITC-conjugated phalloidine (1:200, Yeasen Biotechnology), which was used to label the cytoskeleton. Nuclei were counterstained with DAPI (Abcam, UK).
The optimal cutting temperature (OCT) compound embedded femur was sliced into 10 μm-thick transverse sections. The slices were incubated with primary antibodies against voltage-dependent anion channels (VDAC) (1:1,000, Abcam, UK) at 4°C overnight. The secondary antibody was CY5 fluorescent antibody (1:1,000, Abcam, UK), and the slides were sealed by mounting medium with DAPI. Images were captured with a contained confocal laser scanning microscope system (Olympus).
Briefly, MLOY4 cells were cultured in 35 mm confocal dishes at a density of 2 × 105 cells/dish for 24 hours. They were then fixed in 4% paraformaldehyde for 15 minutes, rinsed with PBS three times, permeabilized with 0.5% Triton X-100 (Beyotime) for 15 minutes, and blocked with 5% goat serum for one hour. The samples were then incubated with the anti-Cx43 rabbit monoclonal antibody (1:1,000, Abcam, UK) overnight at 4°C. The secondary antibody was fluorescein isothiocyanate (FITC) fluorescent antibody (1:1,000, Abcam, UK) and TRITC-conjugated phalloidine (1:200, Yeasen Biotechnology), which was used to label the cytoskeleton. Nuclei were counterstained with DAPI (Abcam, UK).
The optimal cutting temperature (OCT) compound embedded femur was sliced into 10 μm-thick transverse sections. The slices were incubated with primary antibodies against voltage-dependent anion channels (VDAC) (1:1,000, Abcam, UK) at 4°C overnight. The secondary antibody was CY5 fluorescent antibody (1:1,000, Abcam, UK), and the slides were sealed by mounting medium with DAPI. Images were captured with a contained confocal laser scanning microscope system (Olympus).
4
Andrographolide and Bupivacaine Effects on SH-SY5Y Cells
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SH-SY5Y cells were seeded into 24-well plates at a density of 4×105 cells/well overnight. For treatment 1 (Figure 2A ), SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours at 37°C. After that, cells were prefixed in 4% paraformaldehyde at room temperature for 20 minutes and fixed in cold methanol for 10 minutes at −20°C. Later, cells were incubated with primary antibodies for anti-Ki67 (1:1,000;
Abcam, Cambridge, UK) () and DAPI (1:1,000;
Abcam) at 4°C overnight.
For treatment 2 (Figure 5A ), SH-SY5Y cells were incubated with Andro (0 or 200 μM) for 12 hours. Then, the culture medium was changed and cells incubated with 10 nM AZD5363 for 1 hour. Later, the culture medium was changed and cells incubated with Bup (0 or 400 μM) for another 48 hours. Next, cells were incubated with primary antibodies for anti-p-Akt (1:1,000;
Abcam) and DAPI (1:1,000) at 4°C overnight. After that, cells were incubated with goat antirabbit IgG secondary antibodies (1:5,000;
Abcam) at 37°C for 1 hour. Cells were visualized with fluorescence microscopy (Olympus, Tokyo, Japan).
Abcam, Cambridge, UK) () and DAPI (1:1,000;
Abcam) at 4°C overnight.
For treatment 2 (
Abcam) and DAPI (1:1,000) at 4°C overnight. After that, cells were incubated with goat antirabbit IgG secondary antibodies (1:5,000;
Abcam) at 37°C for 1 hour. Cells were visualized with fluorescence microscopy (Olympus, Tokyo, Japan).
5
Immunofluorescence Staining of Stem Cells
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The cells were rinsed with PBS and fixed with 4% paraformaldehyde for 5 min. The cells were permeabilized with 0.05% Triton X-100 at room temperature for 15 min, and washed three times with PBS, and then incubated in 4% goat serum for 30 min. The cells were stained by primary antibodies overnight at 4 degree.The next day, the cells were incubated with Alexa Fluor conjugated secondary antibodies at 37°C for 1 h, and then counterstained with DAPI (Abcam) at room temperature for 5 min, observed under a Leica DMi8 fluorescence microscope (Lecia). To assess pluripotency, hESC colonies were stained with antibody against SOX2 (Abcam), OCT4 (Santa cruz), SSEA4 (Abcam) and NANOG (Santa cruz). To assess differentiated cardiomyocytes, CMs were stained with antibody against cTNT2 (Abcam), α-actinin (Abcam), MLC-2v (Proteintech), TBX18(Proteintech). Secondary antibodies labeled with Alexa Fluor 488 or 594 were applied (Proteintech). Finally, cells were stained with DAPI (Abcam) and Cell area was further quantified by the ImageJ software (National Institutes of Health) as previously described [12] . Information of the used antibodies in this study are listed in Supplementary Table 2.
6
Multimodal Immunofluorescence Microscopy of Tumor Vasculature
7
Cytoskeleton Protein Analysis in Cultured Cells
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Production and arrangement of cytoskeleton proteins were studied during the entire experimental period. Briefly, the cells were detached using trypsin/EDTA (0.25%; Thermo Fisher Scientific), counted using a hemocytometer, and seeded at a density of 10,000 cells/cm2 in gelatin (0.1%; Thermo Fisher Scientific)-coated 12-well plates (Corning) at a density of 10,000 cells/cm2. The day after, the cells were stained for F-actin and the type III intermediate filament protein vimentin. Upon reaching optimal density for imaging, the cells were fixed using a 4% paraformaldehyde solution in DPBS (v/v) (Chem Cruz®, Santa Cruz) as described above. Thereafter, the cells were stained with Alexa Fluor® 488 Phalloidin (1:40 in blocking solution; Thermo Fisher Scientific, cat. no. A12379) and vimentin monoclonal antibody (V9) eFluor 615 (1:500 in blocking solution; Thermo Fisher Scientific, cat. no. 42989782). The cell nuclei were counterstained with DAPI (1:1000 dilution) (BioVision). Confocal images were taken with the microscope Axiovert 200 M microscope (Zeiss, Oberkochen, GE) mounted with LSM 5 Pascal exciter using the LSM 5 Pascal software (Zeiss).
8
Investigating NF-κB Activation in RVECs
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RVECs seeded in 24-well plates were pretreated with/without HCQ for 10 h and incubated with TNF-α for another 24 h at 80%–90% confluency. After stimulation, cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with 3% BSA for 2 h at room temperature. Subsequently, they were stained overnight with rabbit anti-phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, Danvers, Massachusetts, USA) at 4°C, washed with PBS, incubated with Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen, California, USA) for 2 h, and then with DAPI (Abcam, Cambridge, UK) for 10 min. Cells were imaged using an inverted fluorescent microscope (Nikon).
9
Immunofluorescence Staining for LAMP1 and LC3
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Cells were grown on chamber slices (EMD Millipore, Billerica, MA, USA), harvested 3 days after siRNA transfection (described below), fixed in 100% cold methanol, blocked in 10% goat normal serum, 1% BSA and 0.1% Triton X-100/PBS, and incubated with the following antibodies: LAMP1 (dilution, 1:100; cat. no. sc-19992; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and LC3 (dilution, 1:100; cat. no. ab167159; Abcam, Cambridge, MA, USA). Alexa Fluor 488- (1:200; cat. no. ab150073) or Alexa Fluor 594-conjugated secondary antibodies (1:200; cat. no. ab150140; Abcam) were used and DNA was stained with DAPI within the mounting medium (cat. no. ab104139; Abcam).
10
Microglial Activation Quantification Protocol
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HMO6 cells were fixed for 30 min at room temperature in 4% paraformaldehyde in PBS (pH 7.4), and then permeabilized with 0.25% Triton X-100 for 4 min at room temperature. Slides were incubated for 1 h in 10% goat serum/TBS, followed by incubation with 1:100 dilution of mouse anti-Iba1 antibody (Wako Pure Chemical Industries, Ltd.; cat. no. 012-26723) and 1:50 rabbit anti-CD68 (BIOSS, bs-20403R) at 4°C overnight. Then, the sections were incubated with Alexa 568-conjugated donkey anti-rabbit IgG (red) (1:200 in PTwH; Life Technologies; cat. no. A10042) and Alexa 488-conjugated donkey anti-mouse IgG (green; 1:200 in PTwH; Life Technologies; cat. no. A32766) for 2 h at room temperature. Nuclei were stained with DAPI (Abcam). Microgliosis was assessed by measuring the immunoreactivity of Iba1 (expressed by all microglia) and CD68 (expressed by activated microglia). The images were observed under a fluorescence microscope (Olympus IX71; Olympus Corporation; magnifications, ×40 and ×20) and Image Pro-Plus 6.0 software (1993, 2003 Media Cybernetics) was applied to calculate the immunohistochemical optical density.
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