To confirm ChR2 expression (in the primary rat CMs, in the CM/ChR2-HEK co-cultures and the iPS-CMs) and to confirm myocyte-like properties of iPS-CMs, antibody staining and confocal imaging was performed (Fig. 3a,c and Supplementary Fig. 2 ), using the Olympus FluoView FV1000 confocal system. Samples were fixed in 3.7% formaldehyde after performing functional experiments. Before antibody labelling, cell membrane permealization was performed by incubating samples in 0.02% TritonX-100 for 5 min. Cells were labelled with mouse anti-α-actinin primary antibody (Sigma-Aldrich, A-7811) at 1:600 and Alexa Fluor 647 goat anti-mouse IgG secondary antibody (Invitrogen, A21235) at 1:1,000. All antibodies were diluted using 1% bovine serum albumin (Amersham PLC, Amersham, UK). 1% FBS was used as a blocking agent. After antibody staining, cell nuclei were stained with 1 μg ml−1 DAPI with 10 min incubation in PBS. Imaging was done using the Olympus FluoView FV1000 confocal system with acquisition rate at 4 μs per pixel. Gain was kept constant for control and test groups to normalize and exclude autofluorescence contributions.
Alexa fluor 647 goat anti mouse igg secondary antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647 goat anti-mouse IgG secondary antibody is a fluorescently labeled antibody used to detect and visualize mouse primary antibodies in various immunoassays and imaging techniques. The Alexa Fluor 647 dye provides a bright, photostable fluorescent signal that can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview fv1000 confocal system, by Olympus (3 mentions)
Mouse anti α actinin primary antibody, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mouse anti human trop2 antibody, by BD (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Facscanto 2, by BD (1 mentions)
Irdye 680lt goat anti mouse igg, by LI COR (1 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Phospho mlc, by Cell Signaling Technology (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Low endotoxin bsa, by Merck Group (1 mentions)
Pa p9767, by Merck Group (1 mentions)
α catenin, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti rabbit immunoglobulin g igg, by LI COR (1 mentions)
Phospho yap s127, by Abcam (1 mentions)
Y 27632, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
α actinin, by Merck Group (1 mentions)
β catenin, by BD (1 mentions)
Gm130, by BD (1 mentions)
6 protocols using alexa fluor 647 goat anti mouse igg secondary antibody
1
Confirming ChR2 Expression and iPS-CM Properties
2
TROP2 Expression Profiling in Prostate Cancer
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Cells were stained with Mouse anti-human TROP2 antibody (BD Bioscience 551317, 1/200) in antibody staining buffer (0.9% (w/v), sodium azide and 2% FBS in PBS) for 1 hour. Following three washes in PBS, Alexa Fluor® 647 Goat Anti-Mouse IgG secondary antibody (Invitrogen A21236, diluted 1 in 1000 in antibody staining buffer) was incubated for one hour at 4°C. Stained cells were then washed three times in antibody staining buffer prior to FACS sorting and analyzing.
Cell sorting was performed using a MoFlo®AstriosTM (Beckman Coulter). The gating strategy to define negative populations for ALDH and TROP2 staining was designed so as to include at least 99.9% of cells in the DEAB and isotype negative control samples. A distinct population highly expressing TROP2 was identified and sorted from PC3 cells (~2.75% of total viable cells). The same gating strategy was applied to LNCaP, DU145 and 22Rv1 cells. Since no individualized TROPhigh population was detected in these cell lines, the TROP2high population was defined as the top 2% of TROP2-expressing cells. FACS analysis was performed using a FACSCanto™ II (BD Science).
Cell sorting was performed using a MoFlo®AstriosTM (Beckman Coulter). The gating strategy to define negative populations for ALDH and TROP2 staining was designed so as to include at least 99.9% of cells in the DEAB and isotype negative control samples. A distinct population highly expressing TROP2 was identified and sorted from PC3 cells (~2.75% of total viable cells). The same gating strategy was applied to LNCaP, DU145 and 22Rv1 cells. Since no individualized TROPhigh population was detected in these cell lines, the TROP2high population was defined as the top 2% of TROP2-expressing cells. FACS analysis was performed using a FACSCanto™ II (BD Science).
3
Characterizing ChR2 Expression in Cardiac Cells
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Example 7
To confirm ChR2 expression (in the primary rat CMs, in the CM/ChR2-HEK co-cultures, and the iPS-CMs) and to confirm myocyte-like properties of IPS-CMs, antibody staining and confocal imaging was performed (see
4
PSCA-Targeted Antibody Binding Assay
5
Immunoblotting and Immunofluorescence Assay Protocol
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Blebbistatin (B05060), low-endotoxin BSA (A8806), PA (P9767), and Y27632 (Y0503) were from Sigma Aldrich (St. Louis, MO), while cis-PO/palmitoleic acid (10009871) was from Cayman Chemicals (Ann Arbor, MI). Antibodies used for immunoblotting were α-actinin (Millipore Sigma; A7811), β-actin (Cell Signaling; 4790), α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), VE-cadherin (Santa Cruz; 9989), RhoA (Santa Cruz; c-418), IRDye 800CW goat anti-rabbit immunoglobulin G (IgG) (LI-COR; 9253211), and IRDye 680LT goat anti-mouse IgG (92668070). Antibodies and reagents used for immunofluorescence were α-catenin (ThermoFisher; 13-9700), β-catenin (BD Biosciences; 610153), DAPI (4’,6-Diamidino-2-phenylindole, dihydrochloride; ThermoFisher; D1306), GM130 (BD Biosciences; 610823), phospho-MLC (Cell Signaling; 3674), phalloidin (Alexa Fluor 555) (ThermoFisher; A34055), phospho-YAP S127 (Abcam; 76252), YAP (Cell Signaling 14074), VE-cadherin (Santa Cruz; 9989), goat anti-rabbit IgG secondary antibody (Alexa Fluor 488) (ThermoFisher; A11008), goat anti-mouse IgG secondary antibody (Alexa Fluor 647) (ThermoFisher; A21235). Dako fluorescence mounting medium (S3023) was from Aligent. Y227632 (Y0503) was from Millipore Sigma (Burlington, MA). The plasmid encoding GFP–β-actin was kindly provided by Sergio Grinstein (Hospital for Sick Children, Toronto, Ontario, Canada).
6
Immunofluorescence Staining of LRRK2 and Flag
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The cells were plated and grown on a cover glass for the indicated time, washed twice with PBS 1X, and then fixed with 4% paraformaldehyde/PBS1X for 10 min. Cells were permeabilised with 0.1% Triton X-100 diluted in PBS1X. Non-specific binding was blocked with 5% bovine serum albumin (BSA), 0.05% Tween 20 diluted in PBS1X for 1 h at room temperature. Fixed cells were incubated with primary antibodies: anti-LRRK2 (1:500 MJFF2 c41-2 Abcam) and anti-Flag (F3165 1:2000 Sigma-Aldrich), diluted in blocking solution, overnight at 4 °C. Cells were then washed with PBS1X, 0.05% Tween 20 and incubated with secondary antibodies: Goat anti-Mouse IgG Secondary Antibody Alexa Fluor® 488 (Thermo Fisher Scientific) and Goat anti-Mouse IgG Secondary Antibody Alexa Fluor® 647 (Thermo Fisher Scientific), diluted 1:1000 in blocking solution for 1 h at room temperature. Finally, after adding the Mowiol mounting medium, the cells were analysed by Leica TCS SP5 confocal microscope with LAS lite 170 image software (Advance Fluorescence 2.7.3.9723).
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