Fixed cell staining was performed according to the methods described previously [29 ,30 (link)] with minor modifications. Briefly, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 0.2% gelatin for 30 min. The cells were then incubated with primary antibody: mouse monoclonal α-tubulin (1:4,000 dilution, T5168, Sigma-Aldrich) and Alexa Fluor 488 phalloidin (0.33 U/ml, A12379; Life Technologies) for 60 min. After several washes with 0.2% gelatin, the cells were incubated with secondary antibody: Alexa Fluor 546 Anti-mouse IgG (1:2,000 dilution, A-11030, Life Technologies) for 60 min. The fixation and staining were all carried out at room temperature. In the vinculin stain tests, mouse monoclonal vinculin (1:800 dilution, V9131, Sigma-Aldrich) and Alexa Fluor 546 Anti-mouse IgG (1:2,000 dilution, A-11030, Life Technologies) were used as the primary and secondary antibody, respectively. In the myosin IIA stain tests, the cells were permeabilized with 0.02% Triton X-100. Rabbit polyclonal myosin IIA (1:200 dilution, M8064, Sigma-Aldrich) and Alexa Fluor 546 Anti-rabbit IgG (1:2,000 dilution, A-11071, Life Technologies) were then used as primary and secondary antibody, respectively.
Alexafluor 546 anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
AlexaFluor 546 anti-mouse IgG is a fluorescently labeled secondary antibody used in various immunodetection techniques. It binds specifically to mouse immunoglobulin G (IgG) and can be detected under a fluorescence microscope or other fluorescence-based detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Thermo Fisher Scientific (2 mentions)
Hepatocyte growth factor (hgf), by Merck Group (2 mentions)
Glass slides 75 25 mm2, by Avantor (2 mentions)
Collagen from rat tail type 1, by Thermo Fisher Scientific (2 mentions)
Transforming growth factor β1 tgf β1, by Merck Group (2 mentions)
Su11274, by Selleck Chemicals (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Cd117 apc, by BD (1 mentions)
Cd133 pe, by BD (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Mouse anti active β catenin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Filipin, by Santa Cruz Biotechnology (1 mentions)
Hepes, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
14 protocols using alexafluor 546 anti mouse igg
1
Immunofluorescence Staining of Cytoskeletal Proteins
2
Characterization of CSC Phenotype in Spheres
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For further characterization of CSC phenotype of sphere forming SP cells, flow cytometry was used. Spheres were enzymatically dissociated into single cell suspension (1X106). Cells were washed twice with PBS and were stained with CD133-PE and CD117-APC (550412; BD Pharmingen) for 45 minutes at 4°C. Respective mouse IgG isotype were used as control. After incubation cells were washed with PBS and stained with fluorochrome conjugated anti mouse IgG Alexa Fluor 546 (invitrogen) at a dilution of 5 μl of antibody per 106 cell and re incubated further in dark on ice for 30 minute, washed, re-suspended in HBSS and stained by Propidium iodide (1 μg/ml). Analysis and sorting were performed on FACS Aria II using Diva Software.
3
Quantifying OVA and CTB Internalization in Caco-2 Cells
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Caco-2 cells were incubated in glass cover slips for 3 h with OVA Alexa Fluor 647 (50 μg/ml) or CTB Alexa Fluor 647 (10 μg/ml) alone or in presence of different concentrations of U-Omp19 (25 and 50 μg/ml) or Leupeptin (10 μg/ml) in medium. Then, cells were washed, fixed with 2% paraformaldehyde, permeabilized with 0.2% saponin and stained with mouse anti–human Lamp-2 (1:500; BD Biosciences). Secondary antibody anti-mouse IgG Alexa Fluor 546 (Invitrogen) was used. Then, slides were mounted with Aqua-Poly/Mount (Polysciences) and analyzed using an IX-81 microscope attached with a FV-1000 confocal module. For quantification of colocalization Lamp-2/OVA or Lamp-2/CTB, the Manders plugin of ICY Image Software was used and the coefficient was calculated. Single ROIs (Lamp-2+ compartments) were determined automatically with Spot Detector plugin detecting bright spots in dark background using a scale and sensitivity of 7 pixels and size filtering of 50–50,000 pixels. Manders colocalization coefficient was measured in each ROI using Manders plugin.
4
Quantitative Analysis of Active β-Catenin and TNIK
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AML cells were fixed with 4% paraformaldehyde, followed by blocking and permeabilization with 5% goat serum/1% bovine serum albumin in 0.1% PBS–Tween 20 for 1 h in FACS tubes. After washing, cells were incubated for 1 h with mouse anti–active β-catenin (dilution 1:100; clone 8E7; EMD Millipore) and rabbit αTNIK antibody (dilution 1:50; D-16; Santa Cruz Biotechnology, Inc.), followed by incubation with anti–mouse IgG–Alexa Fluor 546 (dilution 1:500; Invitrogen) and goat anti–rabbit IgG–Alexa-Fluor 488 (dilution: 1:1,000; Cell Signaling Technology) for 30 min. Nuclei were stained with 10 µg/ml DAPI. Cells were acquired on an ImageStreamX Mark II imaging flow cytometer (Amnis/EMD Millipore) and analyzed using INSPIRE and IDEAS software (Amnis/EMD Millipore).
5
Intracellular Trafficking of STIP1 Proteins
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The antibodies used in this study were Alexa Fluor 546 anti-mouse IgG, Alexa Fluor 488 anti-mouse IgG, v5 (Life Technologies), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), FLAG (Sigma-Aldrich, St. Louis, MO, USA), STIP1 mouse (Abnova, Taipei, Taiwan), and STIP1 rabbit (Gentex, Taipei, Taiwan). The procedures used for the production and purification of the v5-tagged rhSTIP1 proteins have been previously described in detail.31 (link) The FLAG-Rev peptide consists of a FLAG tag and the Rev peptide, which is a 13-amino-acid fragment of the HIV and has high affinity with the B23 protein.50 (link) The commercial sources of the chemicals were as follows. Sucrose and HEPES were from Sigma, filipin was obtained from Santa Cruz Biotechnology, and amiloride hydrochloride was purchased from Toronto Research Chemicals (Toronto, ON, Canada). CellLight Early Endosomes-GFP BacMan 2.0 (C10586) and CellLight Late Endosome-RFP BacMan 2.0 (C10589) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
6
Immunofluorescent analysis of ECM proteins
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Cells were seeded onto a 4-well Slide & Chamber (Watson, Japan) and incubated at 37 °C for 24 h before fixing immediately in 4% paraformaldehyde and permeabilizing with 0.1% Triton X-100 in PBS for 5 min. The slides were then incubated with the anti-COL1A1 (3G3) antibody (sc-293182, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:50 dilution, v/v), anti-COL3A1 (B-10) antibody (sc-271249, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:50 dilution, v/v), or anti-elastin (BA-4) antibody (sc-58756, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:50 dilution, v/v) at 4 °C overnight, followed by incubation with the secondary antibody, Alexa Fluor® 546 anti-mouse IgG (Life Technologies, Carlsbad, CA, USA, A11060, dilution 1:500) for 30 min at room temperature. Nuclear staining and mounting were performed using VECTASHIELD® Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were captured using a FSX100 fluorescence microscope (Olympus, Tokyo, Japan).
7
Immunostaining Protocol for Troponin-T
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Cells were fixed (4% [v/v] formaldehyde in phosphate-buffered saline [PBS], containing [in mM] NaCl [140], KCl [2.7], Na2HPO4 [10], NaH2PO4 [2], pH 7.4) for 10 min at room temperature (RT) and then washed three times with PBS prior to permeabilization (0.1% [v/v] Triton X-100 in PBS, 4 min at RT). Nonspecific antibody interactions were blocked by incubation in horse serum (4% [v/v] in PBS, 1 h, RT) before cells were incubated with mouse anti–troponin-T (TnT; 1:200 [v/v] in PBS) (MA5-12960; Thermo Scientific, Waltham, MA, USA) overnight at 4 °C. Cells were washed with PBS (3 × 5 min) before being incubated with Alexa Fluor 546 anti–mouse IgG (1:200 [v/v]; Life Technologies) for 1 h at RT in the dark. Following washing with PBS (3 × 5 min), cell nuclei were counterstained with DAPI (1 µg/mL; 20 min) prior to further washing in PBS (2 × 1 min) and mounting under Prolong Gold (Life Technologies). Cells were imaged using a confocal microscope (SP5; Leica Microsystems, Wetzlar, Germany), and assessments of TnT positivity and cellular alignment were made using image analysis (LAS-AF [Leica Microsystems] and ImageJ [National Institutes of Health, Bethesda, MD, USA]).
8
Immunofluorescence Staining of Cells
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Fixed-cell staining was performed according to methods described previously (Miyoshi and Adachi, 2012; Okeyo et al., 2009) , with small modifications. Briefly, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 0.2% gelatin for 30 min. The cells were then incubated with primary antibody, namely mouse monoclonal vinculin (1:800 dilution, V9131, Sigma-Aldrich) or Alexa Fluor 488 phalloidin (0.33 U/mL, A12379; Life Technologies, Carlsbad, CA), or both, for 60 min. After several washes with 0.2% gelatin, the cells were incubated with secondary antibody, namely Alexa Fluor 546 Anti-mouse IgG (1:2,000 dilution, A-11030, Life Technologies), for 60 min. Fixation and staining were all performed at room temperature. For myosin IIA staining, the cells were permeabilized with 0.02% Triton X-100. Rabbit polyclonal myosin IIA (1:200 dilution, M8064, Sigma-Aldrich) and Alexa Fluor 546 Anti-rabbit IgG (1:2,000 dilution, A-11071, Life Technologies) were used as the primary and secondary antibody, respectively.
9
Immunofluorescence Staining of Cultured Cells and Mouse Osteocytes
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For cultured cells, cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, stained using anti-Cas and anti-RelA as primary antibodies as well as Alexa Fluor 488 anti-rabbit immunoglobulin G (IgG) and Alexa Fluor 546 anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) as secondary antibodies, and then viewed with a confocal microscope (Nikon A1R system). 4′,6-Diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) was used to stain the nucleus.
For osteocytes in mouse midshaft tibiae, mice were transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). Tibiae were immersed in the same fixative at 4°C overnight and decalcified in 10% EDTA (pH 7.4) at 4°C for 14 days. The samples were embedded in paraffin, sectioned with 5-μm thickness, and incubated with primary antibodies (anti-Cas, anti-RelA, and anti–acetylated RelA) at 4°C overnight. Alexa Fluor 488–conjugated goat anti-mouse IgG, Alexa Fluor 488 anti-rabbit IgG, and Alexa Fluor 594 anti-mouse IgG were used as secondary antibodies. Nuclei were counterstained using DAPI. Sections were mounted with Fluorescent Mounting Media (Dako, CA, USA). Quantitative 3D analysis of nuclear/total Cas was conducted using Imaris software (Bitplane, Zurich, Switzerland).
For osteocytes in mouse midshaft tibiae, mice were transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). Tibiae were immersed in the same fixative at 4°C overnight and decalcified in 10% EDTA (pH 7.4) at 4°C for 14 days. The samples were embedded in paraffin, sectioned with 5-μm thickness, and incubated with primary antibodies (anti-Cas, anti-RelA, and anti–acetylated RelA) at 4°C overnight. Alexa Fluor 488–conjugated goat anti-mouse IgG, Alexa Fluor 488 anti-rabbit IgG, and Alexa Fluor 594 anti-mouse IgG were used as secondary antibodies. Nuclei were counterstained using DAPI. Sections were mounted with Fluorescent Mounting Media (Dako, CA, USA). Quantitative 3D analysis of nuclear/total Cas was conducted using Imaris software (Bitplane, Zurich, Switzerland).
10
Hepatocyte Culture Optimization Protocol
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