Staining was performed as described for MCF10A acini cultured in Matrigel [75 (link)] using anti E-cadherin (BD Transduction Laboratories) as primary antibody and Alexa-488 conjugated anti-mouse from Molecular Probes as secondary antibody. Nuclei were visualized using DAPI.
Alexa488 conjugated anti mouse
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa488-conjugated anti-mouse is a fluorescently labeled secondary antibody that binds to mouse primary antibodies. It is designed for use in various immunodetection techniques, such as flow cytometry, immunofluorescence, and Western blotting.
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Lab products found in correlation
Lsm 880, by Zeiss (2 mentions)
Anti e cadherin, by BD (1 mentions)
Matrigel, by BD (1 mentions)
Mouse anti flag m2 antibody, by Merck Group (1 mentions)
Latrunculin b, by Merck Group (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fugene hd, by Promega (1 mentions)
Anti rad51 h 92, by Santa Cruz Biotechnology (1 mentions)
Anti flag clone m2, by Merck Group (1 mentions)
Anti β actin clone ac 74, by Merck Group (1 mentions)
Anti stx17 hpa001204, by Merck Group (1 mentions)
Apc conjugated anti human cd69, by BioLegend (1 mentions)
Rabbit anti p70s6k, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
G6767, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
Ezrin, by Abcam (1 mentions)
α catenin, by BD (1 mentions)
7 protocols using alexa488 conjugated anti mouse
1
Immunofluorescent Staining of Acini
2
Visualizing TAZ-CAMTA1 Localization in Cells
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MS1 cells expressing doxycycline-inducible TAZ-CAMTA1 were seeded onto Lab-Tek II removable four-well chamber slides (Thermo Scientific). To induce TAZ-CAMTA1 expression, 2 µg/mL doxycycline were added to the appropriate chambers. After 24 h of seeding, cells were treated with vehicle or the respective drugs. Cells were treated with 1 µM latrunculin B for 1 h (Sigma Aldrich). The primary antibodies used were mouse anti-FLAG M2 antibody (Sigma-Aldrich F1804), Alexa488-conjugated anti-mouse (Molecular Probes), and Alexa fluor 647 phalloidin (Thermo Fisher A22287). For localization quantifications, cells were considered to have either majority nuclear or majority cytoplasmic localization based on immunostaining. The percentages of cells showing nuclear or cytoplasmic staining were averaged over multiple fields of view (n).
For transient transfections inFigure 7 , 293A, 293A LATS1/2 KO, and 293T cells were seeded and transfected with pcDNA3.1 expressing TAZ-CAMTA1 or TAZ-CAMTA1S3A according to the manufacturer's instructions with FuGene HD (Promega). After 48 h of transfection, cells were fixed with 4% PFA in PBS and stained with the appropriate antibodies. Localizations were quantified as above. The individual chambers were removed and slides were imaged with an LSM 880 (Zeiss). Images were postprocessed with Fiji (Fiji is just ImageJ)
For transient transfections in
3
Quantification of DNA Repair Foci in U2OS Cells
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U2OS cells were grown on coverslips and irradiated with 10 Gy 24 h after transfection. Four hours later, the cells were permeabilized by incubating them twice for six minutes on ice in CSK buffer (10 mM PIPES pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, Triton X-100 0.7% and 0.3 mg/mL RNase A) [36 (link)]. From each transfection point, a non-permeabilized control coverslip was directly fixed with 4% paraformaldehyde. The coverslips were incubated with blocking buffer (10% FBS, 1% BSA, 0.2% Triton X-100 in phosphate buffer saline) for 15 min at room temperature, then incubated with the primary antibodies ANTI-FLAG® M2, Clone M2 (1:1,000, F1804, Sigma-Aldrich) and anti-RAD51 H-92 (1:200; Santa Cruz Biotechnology Inc., TX, USA) overnight at 4 °C. After three washes with PBS, the coverslips were incubated with the secondary antibodies Alexa488-conjugated anti-mouse or the Alexa546-conjugated anti-rabbit (Molecular Probes, Eugene, OR, USA). The nuclei were stained with 2 μg/mL DAPI. Images were captured on a Zeiss confocal microscope, at 40× and 63×. The Rad 51 foci were analyzed using ImageJ software (National Institutes of Health (NIH, Bethesda, MA, USA) and Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin, Madison, WI, USA). At least 100 nuclei were counted; cells forming > 5 foci were scored as positive.
4
Immunoblotting and Immunofluorescence Techniques
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Mouse monoclonal anti-LC3 (clone 1703; Cosmo Bio Co., Tokyo, Japan), anti–β-actin (clone AC74; Sigma-Aldrich, Tokyo, Japan), anti-FLAG (clone M2; Sigma-Aldrich), anti-Myc (9E10; Covance Research Products, Denver, PA), anti-EGF receptor (610017; BD Biosciences, Franklin Lakes, NJ), anti-EEA1 (M176-3; MBL, Tokyo, Japan), rat monoclonal anti-GFP antibody (clone GF090R; Nacalai Tesque, Kyoto, Japan), rabbit polyclonal anti-GFP (598; MBL), anti-p62 (PM045; MBL), anti-STX17 (HPA001204; Sigma-Aldrich), anti-VPS33A (C1C3; GeneTex, Irvine, CA), anti-VPS39 (ab107570; Abcam, Cambridge, MA), and anti-UVRAG (A301-996A; Bethyl Laboratories, Montgomery, TX) antibodies were used. The rabbit polyclonal antibodies against LC3 (LC3 #1) for immunoblotting (Hosokawa et al., 2006 (link)) and Atg16L1 (Mizushima et al., 2003 (link)) have been described previously. Rabbit polyclonal anti-LAMP1 antibody was provided by Yoshitaka Tanaka (Kyushu University, Fukuoka, Japan). Rabbit polyclonal anti-VPS16 antibody was provided by Chihiro Akazawa (Tokyo Medical and Dental University; Kim et al., 2001 (link)). Alexa 488–conjugated anti-mouse, rabbit, and rat immunoglobulin G (IgG), Alexa 568–conjugated anti-mouse and rabbit IgG, and Alexa 660–conjugated anti-mouse and rabbit IgG secondary antibodies were purchased from Molecular Probes (Eugene, OR).
5
Immunoblotting and Flow Cytometry Analysis
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The following primary antibodies were used in immunoblotting and flow cytometry: rabbit anti-GAPDH (Santacruz), rabbit anti-phospho-p70 S6 kinase (S6K) (Thr 389) (Cell Signaling), rabbit anti-p70 S6K (Cell Signaling), and APC-conjugated anti-human CD69 (Biolegend). Anti-human CD150 antibody clone 5C6 was generated in our laboratory (Erlenhoefer et al., 2001 (link)). The following labeled secondary antibodies were used: Alexa488-conjugated anti-mouse (Life Technology), Alexa594-conjugated anti-rabbit (Life Technology), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signaling). For flow cytometry 2 × 105 cells per sample were stained with respective antibodies in FACS buffer (PBS containing 0.4% bovine serum albumin (BSA) and 0.02% sodium azide). Dead cells were stained with PI (Biolegend). Cells were acquired immediately using a LSR II flow cytometer (BD) and the data were analyzed using FlowJo (Cytek Development) software.
6
Immunostaining of Mouse Embryos
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Embryos were harvested in PBS, pinned to a silicon‐coated Petri dish and excess tissue trimmed with a blade. For GM130 (1:100, mouse, BD Biosciences #610822) and N‐cadherin (1:100, mouse, BD Biosciences, #610921) antibody staining, fixation was in 4% Paraformaldehyde (PFA) at room temperature for 15 min. For PKCζ (1:100, rabbit, SantaCruz, SC216), PAR3 (1:300, rabbit, Upstate, 07330) and ZO1 (1:50, rat, SantaCruz, SC33725) antibody staining, embryos were fixed in 1% PFA for 15 min at 4°C. Three embryos per marker were used except for ZO1 where two embryos were analysed. After fixation, embryos were permeabilized overnight in PBST (1% Triton‐X100 in PBS) with 0.02% thimerosal and blocked for a day at 4°C with 0.1% BSA (Sigma, A3803‐50G) with 5% heat‐inactivated goat serum (Sigma, G6767) in PBST. Then, embryos were incubated in blocking solution with the desired primary antibody and washed several times overnight in PBST. They were then incubated in the dark for 1 day in blocking solution with the appropriate secondary antibody: Alexa 488‐conjugated anti‐mouse (Life Technologies, A21202), anti‐rabbit (Life Technologies, A11008) or anti‐rat (Life Technologies, A11006) together with 1:2000 ToPro3 nuclear stain (Molecular probes, T3605) and 10 μg/mL RNase A (Sigma, R6513). Finally, embryos were cleared and mounted for imaging (see Supplementary Methods ).
7
Immunostaining Tissue Sections: Antibody Panel
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