ESCs and MSCs were fixed with Bouin’s solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 4 °C and stored. Fixed cells were blocked, permeabilized and incubated with the primary antibodies overnight in TCT buffer (0.25% Carrageenan and 0.1% Tween-20 in Tris-HCl buffer pH 7.8). The primary antibodies used were: Sox2 (1:100, Abcam, Cambridge, UK), E-Cadherin (1:15, BD Biosciences, San Jose, CA, USA), CD44 (1:100, Abcam), βI Integrin (1:100, Abcam), Musashi (1:200, Abcam) and Vimentin (1:100, Abcam). After rinsing, appropriate secondary antibodies conjugated to Alexa fluorophores 488 or 598 (Molecular Probes, Invitrogen, Carlsbad, CA, USA) were diluted at 1:500 in TCT solution and incubated for 1 hr at room temperature. After rinsing, cells were mounted in Vectashield hardset mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector laboratories) to counterstain nuclei. Images were acquired with a Leica DMI 6000B microscope.
Vectashield hardset mounting medium with 4 6 diamidino 2 phenylindole dapi
Manufactured by Vector Laboratories
Sourced in United States
Vectashield hardset mounting medium with 4',6-diamidino-2-phenylindole (DAPI) is a laboratory product designed for use in immunofluorescence and other microscopy techniques. It serves as a mounting medium that hardens to protect fluorescent samples. The product contains DAPI, a fluorescent dye that binds to DNA and can be used to counterstain cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Musashi, by Abcam (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Bouin s solution, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
E cadherin, by BD (1 mentions)
Axiocam camera, by Zeiss (1 mentions)
Observer z1 inverted microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Alexa fluor 568 conjugated donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Dp70 digital camera, by Olympus (1 mentions)
Ax80 microscope, by Olympus (1 mentions)
Streptavidin conjugated to fluorescein, by Vector Laboratories (1 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Rab11a, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Chondroitinase abc, by Merck Group (1 mentions)
Hyaluronidase type 5, by Merck Group (1 mentions)
Tween 20, by BD (1 mentions)
10 protocols using vectashield hardset mounting medium with 4 6 diamidino 2 phenylindole dapi
1
Immunophenotyping of Stem Cells
2
Immunofluorescence Microscopy of Transfected U2OS Cells
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Transfected U2OS cells were plated with poly-L-lysine (Cultrex, cat.# 3438-100-01) coated glass coverslips. Cells were plated at a density of 150 000 cells per well in a 6-well plate and allowed to rest over night. Cells were fixed with 3% paraformaldehyde in PBS for 10–15 min, permeabilized with PBS with 0.5% Triton-X-100 for 10 min and blocked with 5% BSA in PBS for 15 min at room temperature. Primary and secondary antibodies were diluted in 1% BSA in PBS and incubated at 37°C for 1 h. Mounting of the coverslips onto microscope slides was done with Vectashield Hardset Mounting Medium with 4-,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA, USA). Coverslips were visualized and imaged using a Zeiss Observer.Z1 inverted microscope and AxioCam camera with Axiovision software (Carl Zeiss Microscopy GmbH, Jena, Germany).
3
Dual Immunofluorescence for SOX2 and Notch2
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Double fluorescent immunohistochemistry was used to examine the relation between SOX2 and Notch2 in adult tissue. Cryosections were blocked with 2% normal donkey serum for 1 hr at 30°C and incubated in phosphate-buffered saline (PBS) with a primary antibody against goat anti-SOX2 (1:250 dilution, Neuromics, Edina, MN, USA) overnight at 4°C. After washing with PBS, sections were incubated in PBS with a secondary fluorescent antibody, Alexa Fluor 568-conjugated donkey anti-goat IgG (1:200 dilution) (Thermo Fisher Scientific, Waltham, MA, USA), for 30 min at 30°C. After washing with PBS, sections were blocked with 10% normal goat serum and 3% BSA in PBS for 30 min at 30°C and incubated with goat anti-Notch2 antibody (20 μg/ml) (R&D Systems, Inc, Minneapolis, MN, USA) labeled with Alexa Fluor 488 by Zenon® Complex Formation/Labeling reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. Then, fluorescent signals were enhanced with rabbit anti-Alexa Fluor 488 antibody (1 μg/ml) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200 dilution) (Thermo Fisher Scientific). Sections were cover-slipped with Vectashield HardSet mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and observed through an AX80 microscope equipped with a DP70 digital camera (Olympus, Tokyo, Japan).
4
Imaging and Quantification of Lipid Droplets
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Lipid droplets were stained by a specifically lipophilic fluorescent dye as described previously [42 (link),43 (link)]. HepG2 cells were seeded in poly-L-lysine-coated coverslips and treated with vehicle or tanshinone IIA in growth medium or high-fat medium as described above. The cells on coverslips were fixed in cold 4% paraformaldehyde at room temperature for 20 min followed by incubation with 0.5 μM BODIPY493/503 fluorescent dye (Thermo Fisher Scientific) for 15 min. The coverslips were washed three times with PBS and mounted using VECTASHIELD HardSet™ mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). The imaged were observed and photographed with a ZEISS LSM 900 laser confocal microscope (ZEISS Microscopy, Jena, Germany). The mean fluorescent intensities of the lipid droplets per cell were analyzed and quantified in at least 200 cells in 25–30 randomly selected fields (63x) from independent replicates using ZEISS ZEN lite software (ZEN 2.3) (ZEISS Microscopy).
5
Immunofluorescence Staining of Signaling Proteins
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A549 and H1975 cells were cultured on a bio-coated cover slip (BD Bioscience, San Jose, CA, USA) and fixed using 4% paraformaldehyde for 20 min. Subsequently, the cover slips were incubated with the primary antibodies for 15 h at 4 °C. The primary antibodies used were as follows: EGFR (1:300, Santa Cruz Biotechnology); Src (1:300, Cell Signaling Technology); α-tubulin (1:300, Merck Millipore); Rab-11A (1:300, Santa Cruz Biotechnology). After a PBS wash, the cells were incubated with anti-rabbit secondary biotinylated antibody (1:2000, Vector Laboratories, Burlingame, CA, USA) and visualized using streptavidin conjugated to fluorescein (Vector Laboratories, Burlingame, CA, USA). For double immunostaining, the cells were incubated with anti-mouse secondary biotinylated antibody (1:2000, Vector Laboratories, Burlingame, CA, USA) and visualized using streptavidin conjugated to fluorescein (Vector Laboratories, Burlingame, CA, USA). The coverslips were mounted on microscope slides with VECTASHIELD® Hard Set™ mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Confocal fluorescence images were obtained by confocal laser scanning microscopy (LSCM, Nikon A1+, Tokyo, Japan) using an oil immersion lens.
6
Immunohistochemical Evaluation of Human Cells
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The presence of reminiscent human cells was evaluated by fluorescent immunohistochemistry staining targeting human leukocyte antigen (HLA) complexes of human discogenic cells. Sections were deparaffinized and incubated in 0.0005% proteinase (Sigma‐Aldrich, St. Louis, Missouri, USA) tris–HCl (pH 7.6) for 10 minutes at 37°C. Sections were rinsed in PBS and treated with 100 μL 3.0 IU/mL hyaluronidase type V (Sigma‐Aldrich), 0.05 IU/mL chondroitinase‐ABC (Sigma‐Aldrich) PBS for 60 minutes at 37°C. Subsequently, sections were rinsed and blocked with 3% bovine serum albumin (BSA), 0.2% Tween‐20 (Sigma‐Aldrich) PBS for 30 minutes at room temperature. Primary antibody antihuman HLA‐ABC Purified (BD BioScience, Frankilin Lakes, New Jersey, USA) was diluted 1:25 in 1% BSA, 0.2% Tween‐20 PBS, applied and incubated over night at 4°C. Subsequently, samples were rinsed, coated with secondary goat antimouse IgG/IgM Alexa Fluor 488 (Thermo Fisher), and incubated for 1 hour at room temperature. Sections were consecutively rinsed and mounted with VECTASHIELD HardSet Mounting Medium with 4′ 6‐diamidino‐2‐phenylindole (DAPI) (Vector Laboratories, Burligame, California, USA). Finally, all slides were analyzed by LSM 510 META confocal microscope (ZEISS, Oberkochen Germany).
7
Brain Tissue Immunohistochemistry Protocol
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Brain sections were washed in Tris buffer (0.1 M Tris, 0.85% NaCl, pH 7.5) for 5 min. and incubated for 20 min. in 3% H2O2, 10% Methanol in Tris buffer (Sigma-Aldrich). The sections were then washed for 5 min. in Tris buffer, 15 min. in wash buffer (0.1% Triton X-100 in Tris buffer), and 15 min. in blocking buffer (2% BSA, 0.1% Triton X-100 in Tris buffer) (Sigma-Aldrich). Sections were incubated with primary antibody diluted in blocking buffer at room temperature overnight with shaking. Sections were washed for 15 min. in wash buffer and 15 min. in blocking buffer, and incubated for 1 hour at room temperature with secondary antibody diluted in blocking buffer. Sections were washed four times and coverslips were mounted on the slides with VECTASHIELD Hard Set Mounting Medium with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Imaging was performed using an Axioimager. M2 ApoTome.2 microscope equipped with a AxioCam MRm camera (Zeiss, San Diego, CA, USA). Antibodies used are described in Supplementary Table S5 .
8
Rad51 Foci Quantification in Irradiated MEFs
9
Isolation and Characterization of Primary Cells
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Dulbecco's modified Eagle's medium (DMEM), William's E medium, phosphate‐buffered saline (PBS), fetal bovine serum (FBS), epithelial growth factor (EGF), penicillin–streptomycin reagent, Hank's balanced salt solution (HBSS), trypan blue, colcemid, and Sytox® green nucleic acid stain were obtained from Life Technologies (Burlington, Ontario). Corning® Biocoat™ type I collagen‐coated culture dishes and coverslips were obtained from VWR International (Mississauga, Ontario). CIzyme™ collagenase HA and BP protease were obtained from VitaCyte LLP (Indianapolis, Indiana). American Type Culture Collection (ATCC) Eagle's minimum essential medium (EMEM), DMEM, and F‐12K medium were obtained from Cedarlane (Burlington, Ontario). VectaShield hardset mounting medium with 4′,6‐diamidino‐2‐phenylindole (DAPI) was obtained from Vector Laboratories (Burlington, Ontario). Dexamethasone, human insulin, dimethylsufoxide (DMSO), Percoll®, bovine serum albumin (BSA), resorufin ethyl ether, resorufin sodium salt, fluorescamine, ribonuclease (RNase) A, and IGEPAL CA‐630 were obtained from Sigma‐Aldrich Canada Co. (Oakville, Ontario). Bacteriophage lambda cl857 DNA was obtained from Roche Diagnostics (Laval, Quebec).
10
Immunofluorescent Staining of Myogenesis
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Cells were fixed with 4% paraformaldehyde in PBS 5 days after GDF‐8 treatment. Fixed cells were blocked in PBS‐T containing 2% BSA for 1 h, and then incubated overnight at 4 °C with anti‐tenomodulin (Santa Cruz Biotechnology, Dallas, TX, USA) and anti‐myogenin (Abcam) antibodies. Subsequently, cells were stained with Alexa Fluor 488‐ and 588‐conjugated goat anti‐(rabbit IgG) secondary antibody (Thermo Fisher Scientific) at room temperature for 1 h. Cells were then mounted using the Vectashield HardSet Mounting Medium with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).
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