Goat anti rabbit igg h l alexa fluor 488 a 11008

Primary antibodies were as follows: Anti-LC3 (MBL, PM036; Dilution ratio: 1:100 for immunofluorescence microscopy, 1:2000 for western blot.), Anti-LAMP1 (Enzo, ADI-VAM-EN001-F; Dilution ratio: 1:500 for immunofluorescence microscopy.), Anti-β Actin (Huaxingbio, HX1831; Dilution ratio: 1:5000 for western blot.). Secondary antibodies for immunofluorescence microscopy were from Invitrogen as follows: Goat anti-Rabbit IgG (H + L) Alexa Fluor 488: A-11008, Goat anti-Mouse IgG (H + L) Alexa Fluor 488: A-11001, Goat anti-Rabbit IgG (H + L) Alexa Fluor 647: A-21244, Goat anti-Mouse IgG (H + L) Alexa Fluor 647, A-21235). These antibodies were used at 1:500 dilution ratio. Secondary antibodies for western blot were from Huaxingbio as follows: HRP-Goat anti-Mouse IgG(H + L) (HX2032), HRP-Goat anti-Rabbit IgG (H + L) (HX2031). These antibodies were used at 1:5000 dilution ratio. Bafilomycin-A1 was purchased from Cell Signaling Technology (CST 54645S). Protease inhibitor cocktail tablets was purchased from Roche (04693132001).

For staining, cells were cultured in an eight-well chamber. When the cells reached approximately 70–80% confluence, they were fixed in 4% formaldehyde for 24 h, immersed in 0.3% Triton-X100 in Tris-buffered saline for 10 min, and rinsed three times with deionized water for 1 min. Ten drops of blocking buffer immunotech-normal serum were added to each well and allowed to react for 5 min. After removing the serum, the primary antibody, CD31 (ab28364, Abcam, Cambridge, UK, 1:100, 150 μL), was added to the wells and incubated at 4 °C for 24 h. After washing three times, the secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor^® 488 A-11008; Invitrogen, Carlsbad, CA, USA, 1:100), was added and incubated for 1 h. After washing with Tween-20 three times for 10 min each, Vectashield (H-1200 with DAPI) was added to the slide, and the slides were covered. Cells were evaluated using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). CD31 expression was quantified by measuring the percentage area of immunoreactivity using the ImageJ software (NIH, Bethesda, MD, USA).