Briefly, 6-μm-thick sections from the hippoampal DG of the Foxg1 genotype mice (n = 6 in each group) were prepared for immunofluorescence analyses as described previously [12 (link),13 (link)]. To visualize protein expression, sections were first incubated with primary antibodies, including rabbit anti-GFAP (1:200, Wanleibio, cat # WL0836, Shenyang, China), mouse anti-GFAP (1:200, Proteintech, cat# 60190-1-lg, Rosemont, USA), EGFP (1:200, Beyotime, cat # AG281, Shanghai, China), brain lipid binding protein (BLBP) (1:200, Proteintech, cat # 51010-1-AP, Rosemont, USA), Proliferating Cell Nuclear Antigen (PCNA) (1:250, Proteintech, cat # 10205-2-AP, Rosemont, USA), Tbr2 (1:200, Bioss, cat # bs-11331R, Beijing, China), Oligo2 (1:200, Bioss, cat # bs-11194R, Beijing, China), NeuN (1:1200, Bioss, cat # bs-10394R, Beijing, China) and Dcx (1:200, Proteintech, cat # 13925-1-AP, Rosemont, USA). Immunofluorescence secondary antibodies, Dylight 488 (cat # BA1126) and CY3 (cat # BA1032), were obtained from Boster (Wuhan, China). DAPI (KeyGen Biotech, cat # KGA215-50, Shanghai, China) was used to stain the nuclei. After being severally rinsed in PBT, the sections were mounted using antifade mounting medium (Beyotime, Shanghai, China) and fluorescence was visualized using a fluorescence microscope (BX41, Olympus, Tokyo, Japan).
Cyanine3 (cy3)
Manufactured by Boster Bio
Sourced in China
Cy3 is a fluorescent dye commonly used in various biological applications. It exhibits a maximum excitation wavelength of 550 nm and a maximum emission wavelength of 570 nm, making it suitable for a range of fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein isothiocyanate (fitc), by Boster Bio (2 mentions)
Triton x 100, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mouse anti gfap, by Proteintech (1 mentions)
Neuronal nuclei (neun), by Bioss Antibodies (1 mentions)
Dylight 488, by Boster Bio (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Fluoview fv1200, by Olympus (1 mentions)
Dmi8 fluorescent microscope, by Leica camera (1 mentions)
Anti 53bp1, by Abcam (1 mentions)
Anti phospho histone γh2ax, by Merck Group (1 mentions)
Cd133, by Abcam (1 mentions)
Fluorescein isothiocyanate fitc, by Santa Cruz Biotechnology (1 mentions)
Flk 1, by Abcam (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Egm 2mv, by Lonza (1 mentions)
8 protocols using cyanine3 (cy3)
1
Immunofluorescence Analysis of Foxg1 Genotype Mice
2
Quantifying Dendritic Complexity via Sholl Analysis
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Cells were fixed in 4% paraformaldehyde and stained with rabbit anti-MAP2 (Anti-Microtubule-Associated Protein 2) (1:1000, Millipore Bioscience Research Reagents, Bellerica, MA, USA) overnight at 4 °C, followed by incubation with secondary antibodies conjugated with Cy3 (1:200, Boster Bio-Technology, Wuhan, China). Images were collected using an Olympus confocal laser scanning FV1000 microscope (Olympus, Tokyo, Japan). Dendritic complexity was assessed by Sholl analysis [26 (link)]. Here, a series of concentric rings of consistently increasing size are centered over the cell soma and the number of dendritic crossings is measured at each ring. Dendritic arbors with more branches will have an increased number of ring crossings, thus providing a quantitative assessment of dendritic complexity [27 (link)]. The photography and analysis of immunoreactivity with ImageJ software (National Institutes of Health) were carried out in an investigator-blinded manner for the three independent experiments.
3
Osteoclast Differentiation Immunofluorescence Assay
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BMMs were seeded at a density of 1×104 cells/well in 96-well plates and cultured with M-CSF. Twenty-four hours later, RANKL and OB were added into medium. After 3 days, cells were fixed with 4% PFA (20min) and permeabilized with 0.25% Triton X-100 (5min). Osteoclasts were blocked using 2% BSA for 1 hour at room temperature and then incubated using the antibody against p65 or NFATc1 overnight at 4°C. On the next day, secondary antibody conjugated with FITC or Cy3 (Boster, Wuhan) was selected for incubating at room temperature for 1 hour. Actin-Tracker Green was used for marking actin (1hour) and DAPI was used to mark cell nuclei (5min). The fluorescent images were captured by using fluorescence microscope or laser scanning confocal microscope.
4
Immunofluorescence Staining of Hepatocytes
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The cells were cultured onto 15 mm plate and dealt with 260 μmol/L GEN with OA and PA for 18 hours. The dishes were washed with PBS for 3 times and fixed by 4% paraformaldehyde (Solarbio) for 20 minutes. After washed with PBS for 15 minutes, proteinase K (1:1000 with PBS) was added into the dishes for 30 seconds prior to washed by PBS. 1 mL Triton X‐100 (1:1000 with PBS, Beyotime) was added into the cells for 10 minutes. Cells were incubated with the primary antibodies (1:200 with goat serum) overnight at 4°C. The hepatocytes were co‐incubated with secondary antibodies conjugated to Cy3 (Boster) in dark for 45 minutes. The nucleus was stained by DAPI (Solarbio) for 10 minutes. The slides were closed by coverslips and observed under a laser confocal microscopy (Fluoview FV1200, OLYMPUS).
5
Immunofluorescence Quantification of DNA Damage
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After irradiation, the cells on the TW insert membrane (#3460, Corning) were fixed with 4% paraformaldehyde for 15 min, washed with PBS, permeabilized (100 mM TrisCl pH 7.4, 50 mM EDTA, 0.5% Triton100 in H2O) and blocked (3% BSA, 0.1% Tween20, 4xSSC, 7.7 mM NaN3 in H2O). The cells on the membranes were incubated overnight at 4 °C with the primary antibodies anti-phospho histone γH2AX (Millipore, 05-636) and anti-53BP1 (Abcam, ab21083) in PBS. The cells were washed with PBS and incubated with a secondary antibody labeled with Alexa488 (A 2102, Invitrogen) or Cy3 (BA 1034-05, Boster) and DAPI 1 mg/ml (Thermo Scientific) for 90 min at room temperature. The membranes were cut manually and transferred onto slides, mounted with immu-mount and cover-slipped. The γH2AX and 53PB1 foci per nucleus were identified by eye and calculated manually using a Leica DMi8 fluorescent microscope (USA).
6
TGF-β1 and EMT Marker Expression Analysis
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Expression levels of TGF‐β1 and epithelial‐mesenchymal markers in the above HepG2 and BEL 7402 cells with different treatments were detected by immunofluorescence using an avidin‐biotin peroxidase complex method. Briefly, cells were fixed in 4% paraformaldehyde and permeabilized using Triton‐X‐100. Cells were then treated in 3% hydrogen peroxide to inactivate endogenous peroxidase. Non‐specific binding was blocked in PBS containing 10% species‐appropriate normal serum for 1 hour at room temperature. The above primary antibodies for TGF‐β1 (1:100), E‐cadherin (1:200), Cytokeratin18 (1:200), Fibronectin (1:200) and Vimentin (1:200) were applied overnight at 4°C in a humidified chamber. Cells were incubated with the appropriate secondary antibody and visualized by peroxidase substrate in conjunction with FITC or Cy3 (Boster, Wuhan, China).
7
Isolation and Characterization of Murine EPCs
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Using Histopaque 1077 density gradient centrifugation (Sigma-Aldrich, MO, USA), bone marrow mononuclear cells (MNCs) were isolated from C57 mice. MNCs/cm2 (106) were seeded on fibronectin-coated six-well plates in endothelial cell growth medium-2MV (EGM-2MV, Lonza, MD, USA) containing 10% fetal bovine serum, 1% streptomycin and penicillin, and then cultivated in a constant temperature incubator at 37 °C with 5% CO2. The adherent cells were removed 72 h later. Since that, the medium was renewed every 3 days [15 (link)].
After 10 d, adherent cells were fixed with 2.5 mg/mL DiI-acetylated-low density lipoprotein (DiI-ac-LDL, Peking Union-Biology, Beijing, China) for 2 h and with 2% paraformaldehyde (Sigma) for 5 min. Subsequently, cells were incubated with 10 mg/L fluorescein isothiocyanate labeled ulex europaeus agglutinin (Sigma) for 1 h.
EPCs were incubated with primary antibodies CD133 and FLK-1 (both from Abcam, Cambridge, UK) and combined with Cy3 (BOSTER, Wuhan, China) or fluorescein isothiocyanate (FITC; Santa Cruz, CA, USA). Representative micrographs were obtained with a microscope (Olympus, Tokyo, Japan) [16 (link)].
After 10 d, adherent cells were fixed with 2.5 mg/mL DiI-acetylated-low density lipoprotein (DiI-ac-LDL, Peking Union-Biology, Beijing, China) for 2 h and with 2% paraformaldehyde (Sigma) for 5 min. Subsequently, cells were incubated with 10 mg/L fluorescein isothiocyanate labeled ulex europaeus agglutinin (Sigma) for 1 h.
EPCs were incubated with primary antibodies CD133 and FLK-1 (both from Abcam, Cambridge, UK) and combined with Cy3 (BOSTER, Wuhan, China) or fluorescein isothiocyanate (FITC; Santa Cruz, CA, USA). Representative micrographs were obtained with a microscope (Olympus, Tokyo, Japan) [16 (link)].
8
Immunofluorescence Detection of Autophagy
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Immunofluorescence (IF) detection was performed as previously described [58 (link)]. Briefly, UMUC3 and J82 cell transfectants were cultured on cover slides in 10% FBS-supplemented DMEM or MEM in each well of 24-well plates. After synchronization, cells were cultured in complete medium for 12 h, followed by 0.1% FBS medium for another 12 h. The cells were fixed with 4% paraformaldehyde (Sigma Aldrich Corporation, 158127) in PBS (Gibco, 8117296) at room temperature (RT) for 15 min, followed by permeabilization in 0.25% Triton X-100 (Beyotime, ST795) at RT for 15 min and staining with LC3A/B (1:100; Cell Signaling Technology, D3U4 C) for 12 h at 4°C. The slides were washed three times with PBS and stained with 0.1 mg/mL DAPI (Sigma Aldrich Corporation, 9542) and CY3 (1:100; BOSTER, BA1032) for 60 min at RT. The slides were washed three times with PBS and once with ddH2O, and then fixed in glycerin. Cell images were captured using an inverted Nikon fluorescence microscope (A1 R). For the quantification of autophagic cells, puncta were determined in 20 random cells per slide.
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