Cells were grown onto 8 mm diameter coverslips, transfected and fixed in 3.7% formaldehyde solution (47608, Fluka, Loughborough, UK) at room temperature for 15 min. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 and blocked with 10% horse serum to prevent non-specific staining. The primary antibody was then added to the blocking buffer and the cells incubated overnight at 4 °C. Primary antibodies: Mouse anti-γ-catenin (610254, BD Transduction), mouse anti–p120 (P17920, BD Biosciences), mouse anti-α-tubulin (T9026, Sigma-Aldrich), and E-cadherin (ECCD2, Zymed laboratories). The day after, the cells were washed in PBS with 0.1% Triton X-100 for three times and incubated with Alexa 546 goat anti-mouse, Alexa 647 goat anti-mouse, (Molecular Probes, Eugene, OR, USA) fluorophore conjugated secondary antibodies for signal detection. The actin filaments were stained using Alexa 546-conjugated phalloidin (Molecular Probes) added after the blocking step. After washes in PBS with 0.1% triton, coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Prolong Gold Antifade Reagent (Molecular Probes). The micrographs were obtained at Confocal Spectral Leica TCS SP5 and LAS-AF 1.8.1 Leica software.
Confocal spectral tcs sp5
Manufactured by Leica
The Confocal Spectral Leica TCS SP5 is a high-performance confocal microscope system. It is designed for advanced fluorescence imaging and spectral analysis in life science research.
Automatically generated - may contain errors
Lab products found in correlation
Las af 1.8.1, by Leica (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 647, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 546, by Thermo Fisher Scientific (1 mentions)
Alexa 546 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse anti γ catenin, by BD (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Anti γh2ax antibody, by Merck Group (1 mentions)
Formaldehyde solution, by Merck Group (1 mentions)
Vectashield mounting medium for fluorescence, by Vector Laboratories (1 mentions)
2 protocols using confocal spectral tcs sp5
1
Immunofluorescence Staining of Cell Adhesion Proteins
2
Fluorescence Microscopy for Protein Localization
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Protein localization was carried out by fluorescence microscopy. For this purpose, cells were grown on coverslips, transfected and fixed in 3.7% of formaldehyde solution (47 608; Fluka, Sigma-Aldrich Inc Missouri, USA) at room temperature for 15 minutes. After washing with 1x PBS, cells were permeabilized with 0.2% of Triton X-100 in PBS and blocked with 10% of horse serum before overnight incubation with anti-γ-H2A.X antibody, (05-636; Merck Millipore, Thermo Fisher Scientific, Waltham, MA, USA). Finally, cells were washed and incubated with secondary antibodies coupled to fluorescent dyes (Alexa Fluor 488 or/and Alexa Fluor 647). At least 200 cells were quantified in each experimental condition.
For Telomere-induced foci (TIF) evaluation cells were co-immuno stained with anti-TRF1 and anti-γ-H2A.X antibodies. Imaging was carried out at room temperature in Vectashield, mounting medium for fluorescence (Vector Laboratories, Burlingame, USA). Images were acquired with a Confocal Spectral Leica TCS SP5. Microscope using an HCX PL APO Lambda blue 6361.40 OIL UV, zoom 2.3 lens using LAS-AF 1.8.1 Leica software and processed using LAS-AF 1.8.1 Leica software and Adobe Photoshop CS. Colocalization of TRF1 and γ-H2A.X was quantified in at least 200 cells in each condition.
For Telomere-induced foci (TIF) evaluation cells were co-immuno stained with anti-TRF1 and anti-γ-H2A.X antibodies. Imaging was carried out at room temperature in Vectashield, mounting medium for fluorescence (Vector Laboratories, Burlingame, USA). Images were acquired with a Confocal Spectral Leica TCS SP5. Microscope using an HCX PL APO Lambda blue 6361.40 OIL UV, zoom 2.3 lens using LAS-AF 1.8.1 Leica software and processed using LAS-AF 1.8.1 Leica software and Adobe Photoshop CS. Colocalization of TRF1 and γ-H2A.X was quantified in at least 200 cells in each condition.
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