Cultured U87, U87 EGFRvIII, LN229, and U251 cells were fixed with 4% paraformaldehyde in PBS (Santa Cruz Biotechnology, USA) for 20 min at room temperature. Thereafter, the cells were permeabilized with 0.2% Triton X 100 in PBS for 15 min. Non-specific binding was blocked by incubation with 5% goat serum in PBS for 30 min. The following primary antibodies used for immunofluorescence: anti-HOXA13 antibody (Abcam, UK; dilution 1:100), anti-EGFR antibody (Invitrogen, USA; 2 μg/ml), anti-phosphpo-SMAD2 antibody (Invitrogen, USA; 2 μg/ml), anti-SMAD3 antibody (Invitrogen, USA; at concentration of 2 μg/ml), anti-SMAD2 antibody (Invitrogen, USA; 2 μg/ml), and anti-phosphpo-SMAD3 antibody (Abcam, UK; dilution 1:100). The slides were then incubated in the appropriate antibodies in antibody dilution buffer overnight at 4°C, followed by a further incubation at room temperature for 1 h with Alexa Fluor®488 donkey anti-mouse IgG antibody (Invitrogen, USA; 1:500), Anti-rabbit IgG Fab2 Alexa Fluor®488 antibody (Cell Signaling Technology, USA; dilution 1:500) or Alexa Fluor®594 donkey anti-rabbit IgG antibody (Invitrogen, USA; 1:500). The samples were washed and embedded into mounting medium with 4, 6-diamino-2-phenylindole (DAPI; nuclear DNA was labeled in blue; VECTOR, USA). Microscopy analysis was performed using a confocal laser scanner microscope.
4 6 diamino 2 phenyl indole dapi
Manufactured by Vector Laboratories
Sourced in United States
4',6-diamidino-2-phenylindole (DAPI) is a fluorescent dye used to stain cell nuclei. It binds to the minor groove of double-stranded DNA, emitting blue fluorescence when excited by ultraviolet light. DAPI is commonly used in microscopy and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Anti hoxa13 antibody, by Abcam (1 mentions)
Anti egfr antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg fab2 alexa fluor 488 antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 donkey anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Prb 160p, by Fortrea (1 mentions)
Prb 165p, by Fortrea (1 mentions)
Prb 145p, by Fortrea (1 mentions)
Ab52987, by Abcam (1 mentions)
Ab31851, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Bx51 epifluorescence microscope, by Olympus (1 mentions)
Ab41803, by Abcam (1 mentions)
15 protocols using 4 6 diamino 2 phenyl indole dapi
1
Immunofluorescence Analysis of Glioma Cell Lines
2
Immunostaining of PLA2G2F in Tissue
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5-µm-thick mouse or human tissue sections were incubated with 1x Blockace (DS Pharma BioMedical) in PBS-T for 30 min, washed three times with PBS-T for 5 min each, and incubated with rabbit anti–mouse or –human PLA2G2F antibody (Degousee et al., 2002 (link)) at 1:500–1,000 dilution in a 10-fold-diluted Blockace for overnight at 4°C. The sections were then washed 3 times with PBS-T for 5 min each time and incubated with Alexa Fluor 647–labeled goat anti–rabbit IgG antibody (Molecular Probes; 1:1,000) at 20°C for 1 h. For double immunostaining, the sections were washed three times with PBS-T for 5 min each and incubated with rabbit antibodies against mouse loricrin, cytokeratin 1 and cytokeratin 5 (PRB-145P, PRB-165P and PRB-160P [Covance], respectively; 1:500) prelabeled with Alexa Fluor 555 (Zenon Labeling System; Molecular Probes) at 20°C for 1 h. Counterstaining was performed with 4,6-diamino-2-phenylindole (DAPI; Vector Laboratories). Stained sections were analyzed with a confocal laser-scanning microscope (LSM510 META, Carl Zeiss). Human skin sections were obtained by surgery at Chiba University (Chiba, Japan) after approval by the Faculty ethics committee and informed consents from patients.
3
Immunofluorescence Analysis of Peripheral Nerve
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Teased nerve fibers mounted on slides were treated with PBS containing 4% Paraformaldehyde for 30 min and blocked with PBS containing 0.2% Triton X-100 and 2% BSA for 60 min. Nerve fibers were incubated with primary antibody (anti-p75NTR antibody 1:1000, ab52987, Abcam; anti-myelin protein zero (MPZ) antibody, 1:1000, ab31851, Abcam; anti-c-JUN antibody, 1:1000, #9165, Cell Signaling; anti-Ki67 antibody,1:100, ab16667, Abcam) for 16 h at 4°C and washed three times with PBS. Next, slides were incubated with Alexa 549- or 488-conjugated secondary antibody (1:800, Alexa Fluor) for 2 h at room temperature and washed three times with PBS. Finally, slides were incubated with PBS counterstained with 4′6-diamino-2-phenyl indole (DAPI; Vectashield, Vector Laboratories) to visualize nuclei. DAPI staining was used for enumeration and identification of nuclei. The slides were visualized using a 20×/0.50 Plan-Neofluar lens (Carl Zeiss). c-JUN-positive endonuclear cells were counted to analyze percent of c-JUN (+)in three independent experiments. Ki67-positive endonuclear cells were counted to analyze percent of Ki67 (+)in three independent experiments.
4
Pollen Viability and Growth Analysis
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Images of seed sets were recorded after dissection of at least 30 siliques from 7-week-old plants under a Lumar V12 fluorescence stereomicroscope (Zeiss, http://microscopy.zeiss.com/microscopy ) connected to an AxioCam MRc5 CCD unit. In vitro pollen germination was performed according to Lin et al. (2014 (link)). Pollen was incubated in the dark for 4 h at 25 °C, then observed under a light microscope. Pollen was collected from at least three different plants, and 100 pollen grains were used to estimate pollen viability, with three replicates. Analysis of in vivo pollen-tube growth was as described (Szumlanski and Nielsen 2009 (link); Lin et al. 2014 (link)). Emasculated pistils from (a) WT, (b) single homozygous bet11-1 and bet12-1, (c) bet11/− bet12/+ and (d) bet11/+ bet12/− were cross-pollinated and collected after 48 h. Mature pollen morphology was analyzed by staining with 1.5 mg/mL 4′, 6-diamino-2-phenylindole (DAPI) (Vector Laboratories, http://www.vectorlabs.com ) solution for 15 min in the dark, then observed under an Olympus BX51 epifluorescence microscope coupled to an Olympus DP70 CCD unit (Olympus, http://www.olympus-global.com/en/corc/company/lifescience ).
5
Immunofluorescence Imaging of AnxA2 in PC12 Cells
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PC12 cells were grown on poly-L-Lys-coated glass coverslips and treated as indicated above. The cells were fixed, permeabilised and blocked against non-specific binding of antibodies as described previously (Grindheim et al., 2014 (link); Grindheim et al., 2016 (link)) prior to staining with primary polyclonal antibodies against AnxA2 (1:250; ab41803, Abcam, Cambridge, UK). The bound primary antibodies were detected using appropriate DyLight-488- or DyLight-594-conjugated Fab2 fragments (1:50; Jackson ImmunoResearch Laboratories, West Grove, United States). The coverslips were inverted and mounted on objective glasses on a small drop of Vectashield mounting medium containing 4′,6-diamino-2′-phenylindole (DAPI) (Vector Laboratories, Burlingame, United States). Confocal imaging was performed using a Leica SP5 AOBS confocal laser scanning microscope equipped with 405 diode and argon and helium neon lasers (Leica Microsystems, Wetzlar, Germany). Optical sections were obtained using the 63×/1.4 NA HCX Plan-Apochromat oil-immersion objective (Leica, Wetzlar, Germany), ∼1 Airy unit pinhole aperture and appropriate filter combinations. Confocal images were obtained in Leica Application Suite (LAS) AF. Figures were made in Microsoft Publisher for the images and GraphPad Prism for the graphs. Quantitation was done with ImageJ and transferred to GraphPad.
6
Immunofluorescence and FISH Protocols
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Immunofluorescence and FISH were performed by modifying existing protocols (49 (link)). Briefly, cells grown on glass coverslips were fixed with 4% paraformadehyde in PBS for 10 min, permeabilized in PBS containing 0.5% Triton X-100 for 10 min, and then blocked with PBS containing 0.5% bovine serum albumin and 0.2% cold fish gelatin for 10 min. Cells were then incubated with mouse anti-TIN2, mouse anti-Myc, and rabbit anti-TOM20 for 16 h at 4°C. After thorough washing with PBS, cells were incubated with Alexa Fluor 488-conjugated anti-rabbit immunoglobulin and Alexa Fluor 568-conjugated anti-mouse immunoglobulin (Molecular Probes). For TIF analysis, cells were incubated with rabbit anti-53BP1 (sc-22760, Santa Cruz Biotechnology) or rabbit anti-γH2AX (2577, Cell Signaling Technology), followed by incubation with Alexa Fluor 488-conjugated anti-rabbit immunoglobulin. Telomere FISH staining was performed with Cy3-(CCCTAA)3 peptide nucleic acid probe (Panagene) as previously described (50 (link)). Cells were counterstained with 4,6-diamino-2-phenylindole (DAPI) (Vectashield; Vector Laboratories). Immunofluorescence images were captured using a confocal laser-scanning microscope LSM 700 (Carl Zeiss).
7
Quantifying Apoptosis in Lung Tissue
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End labeling of the exposed 3′-OH ends of DNA fragments in paraffin-embedded lung tissue was performed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer’s instructions. Cell nuclei were counterstained with VECTASHIELD mounting medium containing 4′,6-diamino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were observed under a laser-scanning confocal microscope (LSM-880, Carl Zeiss, Germany). After staining, the number of TUNEL-positive cells (apoptotic cells) was evaluated in ten random fields per mouse. The percentage of TUNEL-positive cells among the total nuclei was computed for each image, and a mean value was obtained for each mouse.
8
Multicolor Immunofluorescence Staining Protocol
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Immunofluorescence multiple-labeled staining was carried out using a tyramide signal amplification (TSA)-Kit (PerkinElmer Life Sciences, Massachusetts, USA). Firstly, the sections were incubated with the primary antibody p-JNK overnight at 4℃. Then, the sections were incubated with the HRP-labeled secondary antibody for 50 min after cleaning. At last, the sections were incubated with fluorescein isothiocyanate isomer I (FITC)-TSA for 10 min after cleaning. Antigen retrieval was performed by heating in a microwave. The sections were then incubated with the primary antibody YAP overnight at 4℃ followed by incubation of the HRP-labeled secondary antibody for 50 min after cleaning. After that, the sections were incubated with 647-TSA for 10 min after cleaning. Antigen retrieval was again performed by heating in a microwave. Next, the sections were incubated with the primary antibody LC3B overnight at 4℃. Then, the sections were again incubated with the Cyanine3 (Cy3)-labeled fluorescent secondary antibody for 50 min after cleaning. Finally, the sections were incubated with 4,6-diamino-2-phenyl indole (DAPI, 1:500 dilution; Vector Laboratories, CA, USA), and observed under fluorescence microscope.
9
Immunofluorescence Staining of Rat Bladder
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The rats were sacrificed after 1, 2, and 4 weeks, and the sectioned bladders were deparaffinized, rehydrated, treated with 3% hydrogen peroxide to block endogenous peroxidase, rinsed, and then kept in 0.01 M PBS. Next these sectioned bladders were microwaved to retrieve the antigen and then exposed to a 10% normal serum to block any nonspecific reactions. Then, the sections were incubated at 4°C overnight with anti-MIF antibody (diluted 1 : 200; Abcam, Cambridge, UK) and anti-macro antibody (diluted 1 : 200; Abcam, Cambridge, UK) for MIF/Macro costaining. After washing with PBTx, the samples were then incubated with secondary antibody [Alexa Fluor 568 goat anti-rabbit IgG; Alexa Fluor 488 goat anti-mouse IgG Invitrogen, Carlsbad, CA, USA] in dilute solution at room temperature for 1 h. After washing with PBTx, a coverslip was mounted on the slide using mounting medium with 4,6-diamino-2-phenyl-indole (DAPI; Vector Labs Burlingame, CA, USA) to observe the cell nuclei. Digital images were obtained using an Olympus BX51 fluorescence microscope.
10
Characterization of Human Periodontal Ligament Cells
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To determine the source and characteristic of HPDLCs, immunofluorescence detection was performed on the HPDLCs at 3rd passage. Preliminary experimental analysis showed that 5000 cells per well plate in a 24-well plate were optimal for cell identification. Briefly, 1.5 mL DMEM medium containing 10% FBS was added into the cells, which were washed twice by PBS buffer, fixed with 4% paraformaldehyde for 30 min at room temperature and blocked by human mesenchymal stem cell characterization kit (Millipore, Billerica, MA, USA). Next, vimentin and cell keratin (mouse, vimentin BM0135, cytokeratin BM0030, 1:200, BosterBio, Wuhan, China) were added into the cells and held overnight at 4 °C. After being rinsed in PBS buffer for 3 times, the cells were incubated with fluorescein isothiocyanate and secondary antibody horseradish peroxidase-conjugated goat anti-mouse IgG (A0216, 1:500, Beyotime, Suzhou, China) at room temperature for 45 min [19 (link)]. Then, the cells were redyed with 4, 6-diamino-2-phenylindole (DAPi, 1:100, Vector Laboratories, Burlingame, CA, USA). Fluorescence microscopy (BX-41, Olympus Optical, Tokyo, Japan) was used for image analysis.
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