Alexa fluor 568 goat anti mouse secondary

The following antibodies were used for immunofluorescence (IF): anti-E-cadherin (1:100; Cell Signaling), anti-β-catenin (1:100; Sigma-Aldrich), anti-Lamp1 (1:100; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; Life Technologies). IF was performed as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek) and were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was performed using Volocity software (PerkinElmer).

Immunofluorescence was performed on cells cultured on glass‐bottom dishes (P35G‐1.5‐20‐C, MatTek, Ashland, MA, USA), as described previously.⁷ Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20℃, followed by three 5‐min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 h, followed by incubation with primary antibodies at 4℃ overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (D1306, Life Technologies, Carlsbad, CA, USA). Confocal microscopy was performed with the Ultraview Vox spinning‐disk confocal system (PerkinElmer) equipped with a Yokogawa CSU‐X1 spinning disk head and an electron‐multiplying charge‐coupled device camera (Hamamatsu C9100‐13) coupled to a Nikon Ti‐E microsope; image analysis was done using Volocity software (PerkinElmer). The following antibodies were used for immunofluorescence: anti‐phospho‐mTOR (Ser2448) (5536, Cell Signaling, Beverly, MA, USA), anti‐LAMP1 (555798, BD Biosciences, San Jose, CA, USA), anti‐BrdU (5292, Cell Signaling, Beverly, MA, USA), Alexa Fluor 568 goat anti‐mouse secondary (A‐11031, Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 488 goat anti‐rabbit secondary (A‐11034, Life Technologies, Carlsbad, CA, USA).

The following antibodies were used for immunofluorescence (IF): anti-β-catenin (1:100; C2206; Sigma-Aldrich), anti-Lamp1 (1:100; 555798; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; A-11031; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; A-11034; Life Technologies). IF was performed on cells cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek), as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; D1306; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was done using Volocity software (PerkinElmer).