To examine the patterning of PMCs, embryos were immunolabeled using the 1D5 antibody (a kind gift from Dr. David McClay, Duke University) as previously described (McClay et al., 1983 ) with minor modifications. After microinjection, embryos were fixed overnight at 4 °C in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in filtered ASW. After washing for 10 min three times in PBS-Tween (PBS containing 0.05% Tween-20 [Bio-Rad, Hercules, CA]), embryos were blocked in 4% sheep serum in PBS-Tween for 1 h at room temperature. Embryos were incubated in 1D5 antibody at 1:100 dilution in 4% sheep serum in PBS-Tween at 4 °C for 36–48 h. They were then washed three times in PBS-Tween and incubated in Alexa 488 conjugated goat-anti-mouse antibody (ThermoFisher, Waltham, MA) at a 1:300 dilution in 4% sheep serum in PBS-Tween for 1 h at room temperature. After washing three times in PBS-Tween, they were counterstained with Hoechst 33342 (Lonza, Basel, Switzerland) at 1:1000 dilution in PBS-Tween for 5 min and washed three more times with PBS-Tween. Embryos were then imaged using Zen software with a Zeiss LSM 880 confocal microscope, or AxioVision software with a Zeiss Observer Z1 fluorescent microscope (Zeiss, White Plains, NY).
Hoechst 33342
Manufactured by Lonza
Sourced in United States, Switzerland
Hoechst 33342 is a fluorescent dye that binds to DNA. It is commonly used in cell and molecular biology applications to stain and visualize nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
X tremegene 9 reagent, by Roche (2 mentions)
Alexa 488 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Observer z1 fluorescent microscope, by Zeiss (1 mentions)
Pbs tween, by Bio-Rad (1 mentions)
Pacgfp1 n1 vector, by Takara Bio (1 mentions)
Mitotracker red cmxros, by Lonza (1 mentions)
Acldl, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by PromoCell (1 mentions)
Basic fibroblast growth factor (bfgf), by PromoCell (1 mentions)
Fetal bovine serum (fbs), by PromoCell (1 mentions)
Insulin like growth factor 1, by PromoCell (1 mentions)
A1r laser confocal microscope, by Nikon (1 mentions)
Alexa fluor 555 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Verapamil, by Merck Group (1 mentions)
Pacgfp1 n1, by Takara Bio (1 mentions)
Triton x 100, by Merck Group (1 mentions)
18 protocols using hoechst 33342
1
Immunolabeling of Pluteus Larvae for PMC Patterning
2
Subcellular Localization of GFP-Tagged Proteins
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Expression of GFP-fused protein and analyses of its subcellular localization were performed essentially as described previously [23 (link)]. Briefly, expression constructs were prepared by inserting coding sequences of candidate proteins into the pAcGFP1-N1 vector (Clontech) to fuse GFP at the C-terminus of target proteins. The constructs were transfected to human HEp-2 or HEK293T cells using X-tremeGENE 9 reagent (Roche). The parental cell lines were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. After 24-h incubation, nuclear DNA and mitochondria were stained with Hoechst 33342 (Lonza) and MitoTracker Red CMXRos (Lonza), respectively. Fluorescent images were obtained using a confocal microscope, LSM710 (Carl Zeiss).
3
Isolation and Characterization of EPCs
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PBMNCs were incubated for 7 days on fibronectin-coated dishes using endothelial cell growth medium MV2 supplemented with 10% fetal bovine serum, hydrocortisone, ascorbic acid, heparin sulfate, 2% penicillin/streptomycin, 50 ng/mL human recombinant VEGF, insulin-like growth factor 1, basic fibroblast growth factor, and epidermal growth factor (all material from PromoCell). To confirm the cultured cells as EPC, we fixed them with 4% paraformaldehyde and stained them using FITC-conjugated Ulex europeaus agglutinin-I (UEA-1, Sigma). Additionally, the uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine (DiI)-labeled acetylated low-density lipoprotein (acLDL, Life Technologies) was monitored, as cultured-EPCs are known to be positive for both UEA-1 and acLDL. Double-positive cells that adhered to the fibronectin-coated dishes were counted in five randomly selected fields using an inverted fluorescent microscope. Nuclei were visualized using Hoechst 33342 (Lonza). To assess the adhesion ability of the cultured-EPCs, the number of adherent EPCs was compared to the initial number of PBMNCs plated.
4
TRAP Immunofluorescence Assay for Osteoclast-like Cells
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TRAP immunofluorescence assay was performed on FFPE sections, deparaffinised and rehydrated following the procedure described above. For antigen detection the sections were incubated with mouse monoclonal antibody Tartrate Resistant Acid Phosphatase (26E5) MA5-12387 (Thermo Fisher Scientific) followed by the incubation with the secondary antibody Alexa Fluor 488 goat anti-mouse IgG. The nuclei were counterstained with Hoechst33342 (Lonza). Finally, they were analysed using a Nikon's A1R Confocal laser microscope. NIS Elements software was used to perform OCL-like cells and nuclei counts on GCT and GCT/PDB tumor biopsies, randomly selected. TRAP-positive cells with more than 3 nuclei per cell were considered multinucleated OCL-like giant cells. The counting was performed using three 20x microphotographs derived from each sample and calculated as the mean count in the 3 microphotographs of several biopsies carrying the same mutation.
5
Immunohistochemistry of Tadpole Tail
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Immunohistochemistry was performed essentially as described previously29 . In brief, tadpole tails were amputated at 5 dpf (stage 39–41) and fixed at 3 dpa (stage 47) with MEMFA fixative, embedded with 25% gelatin 15% sucrose, frozen, sagittally sliced at 10 μm thick, and incubated with anti-phospho-Stat3 (Tyr705) mouse antibody (Cell Signalling Technology, MA, #9138S) following incubation with Alexa Fluor 555-conjugated anti-mouse IgG goat antibody (Invitrogen, A-21424). Nuclei were counterstained with 10 μg/ml of Hoechst 33342 (Lonza, Switzerland).
6
Side Population Analysis of Cells
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Side population analysis was carried out as described previously.16 Briefly, the cells were labeled with Hoechst 33342 (Lonza, Walkersville, MD, USA) dye for 90 min at a concentration of 2.5 μg/mL with or without Verapamil (Sigma‐Aldrich), which is an inhibitor of ABC transporters, at concentrations of 100 μmol/L. The cells were counterstained with 1 μg/mL PI (Sigma‐Aldrich) for labeling dead cells. Analyses and sorting were carried out with a FACSAria II cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA).
7
Expression and Visualization of Dsup Protein
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For expression of GFP-fused full-length Dsup protein, the coding sequence of Dsup was amplified and inserted into Asp718 and BamHI sites of pAcGFP1-N1 (Clontech). HEK293T cells were transiently transfected with the expression construct using X-tremeGENE 9 reagent (Roche). After 24 h, the cells were stained with Hoechst 33342 (Lonza) to visualize nuclear DNA. Fluorescent signals were observed under a confocal microscope (LSM710, Carl Zeiss).
8
Multimarker Immunofluorescence Staining Protocol
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Samples were washed with PBS, fixed with 4% paraformaldehyde solution (Wako Pure Chemical Industries) for 20 min, permeabilized with 1% Triton™ X-100 (Sigma-Aldrich) in PBS for 20 min, and then stained overnight with the following antibodies: phycoerythrin (PE) mouse anti-human NANOG, Alexa Fluor® 488 mouse anti-OCT3/4 (BD Biosciences), mouse anti-TUJ1 (β-tubulin III; Sigma-Aldrich), rabbit anti-α-SMA (PA1-37024, Thermo Fisher Scientific), and rabbit anti-AFP (Abcam, Cambridge, UK). The secondary antibodies Alexa Fluor® 488 goat anti-mouse IgG (Thermo Fisher Scientific) and Alexa Fluor® 568 Goat Anti-Rabbit IgG (Thermo Fisher Scientific) were then added. The nuclei were stained with Hoechst 33342 (Lonza).
9
Trehalose-Mediated Hyperosmotic Assay
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As a hyperosmotic treatment, the S2 cells were exposed to the culture medium
containing 0.4 M trehalose for 48 h. The cells were stained with Hoechst33342
(6.7 μg/mL, Lonza) and PI (0.67 μg/mL, Dojindo) for 30 min and observed with a
fluorescence microscope BZ-X810 (Keyence). Hoechst33342-positive and PI-negative
cells were counted as live cells and double-positive cells were counted as dead
cells. The survival rates were calculated in 6 wells for each condition by
counting 500 to 700 cells/well using analysis software Hybrid Cell Count
(Keyence).
containing 0.4 M trehalose for 48 h. The cells were stained with Hoechst33342
(6.7 μg/mL, Lonza) and PI (0.67 μg/mL, Dojindo) for 30 min and observed with a
fluorescence microscope BZ-X810 (Keyence). Hoechst33342-positive and PI-negative
cells were counted as live cells and double-positive cells were counted as dead
cells. The survival rates were calculated in 6 wells for each condition by
counting 500 to 700 cells/well using analysis software Hybrid Cell Count
(Keyence).
10
Peptide Synthesis and Cytotoxicity Evaluation
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The 2-chlorotrityl chloride resin, 1-hydroxybenzotriazole (HOBt) O-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU), and N-fluorenyl-9-methoxycarbonyl (Fmoc) protected L-amino acids (Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH) and Fmoc-Lys(Fmoc)-OH) were obtained from GL Biochem. Ltd. Diisopropylethylamine, N,N-dimethylformamide (DMF), piperidine, anhydrous ether, thioanisole, ethanedithiol, trifluoroacetic acid, and dimethylsulfoxide (DMSO) were purchased from Shanghai Chemical Co. IR780 was obtained from Sigma-Aldrich LLC. All other reagents were analytical grade and used as received.
Fetal bovine serum (FBS), Roswell Park Memorial Institute medium (RPMI) 1640, minimum essential medium (MEM), penicillin-streptomycin, and phosphate-buffered saline (PBS) were obtained from Shanghai XP Biomed Ltd. Hoechst 33342 was purchased from Lonza Group Ltd. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Innochem Ltd. An apoptosis and necrosis assay kit and Calcein AM were obtained from Beyotime Biotechnology.
Fetal bovine serum (FBS), Roswell Park Memorial Institute medium (RPMI) 1640, minimum essential medium (MEM), penicillin-streptomycin, and phosphate-buffered saline (PBS) were obtained from Shanghai XP Biomed Ltd. Hoechst 33342 was purchased from Lonza Group Ltd. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Innochem Ltd. An apoptosis and necrosis assay kit and Calcein AM were obtained from Beyotime Biotechnology.
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