Alexa fluor 494 goat anti mouse

For immunocytochemistry, HEK293 cells were seeded on 0.1 mg/ml poly-L-lysine-coated (PLL) coverslips and incubated for 36 hrs. After fixation for 10 min with 4% paraformaldehyde in 0.2 M phosphate buffer and three washes in PBS, cells were incubated with blocking solution (0.3% Triton X-100, 3% bovine serum albumin, 10% goat serum in PBS) for 30 min. All steps were performed at room temperature. Primary antibody solution (mouse anti-N1/12, recognizing KCC2 (Biolegend, San Diego, USA, dilution 1:1,000) or mouse anti-HA.11 (Biolegend, San Diego, USA, dilution 1:250) [46 (link)] were added in blocking solution for 30 min. After three wash steps with PBS for 5 min, a secondary antibody, conjugated to a fluorescent probe (1:1000; Alexa Fluor 494 goat anti-mouse (Invitrogen), was added. After washing, cells were mounted onto glass slides with Vectashield Hard Set (Vector laboratories, Burlingame, CA). Photomicrographs were taken using a Laser scanning microscope (Leica TCS SP2).

Cells were fixed on glass slides with methanol 20%(v/v), embedded in HCL 2M for 30 minutes at RT, incubated with anti-BrdU (1:10) or anti-CXCR2 (1:200) (Abcam) for 60 minutes at RT, then incubated with Alexa Fluor 494 goat anti-mouse or Alexa Fluor 494 goat anti-rabbit secondary antibodies (1:100) (Invitrogen) for 60 minutes, at RT, in the dark. Cells nuclei were stained with DAPI (1:10) (Thermo Fisher) for 15 minutes at RT. Images were acquired in Zeiss Z1 apotome microscope (LEICA) at 10x magnification.