Alexa fluor 488 labeled anti mouse antibody

AN3CA, Nestin knockdown AN3CA, KLE, Nestin knockdown KLE, Ishikawa, and Nestin overexpressing Ishikawa cells endometrial cancer cells seeded on coverslips were fixed in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with phosphate buffered saline (PBS) containing 0.1% Triton X-100 (PBS-T) for 10 minutes, and subsequently blocked with PBS-T containing 5% normal goat serum for 1h. Cells were stained with mouse monoclonal anti-cortactin antibody (EMD Millipore) for 1h in PBS-T containing 1% normal goat serum and a secondary antibody Alexa Fluor^® 488-labeled anti-mouse antibody (Invitrogen Life Technologies). To detect F-actin, cells were stained with Phalloidin–Tetramethylrhodamine B isothiocyanate (Sigma-Aldrich) for 20 minutes and the cell nuclei were stained with 1 μg/mL DAPI for 10 minutes. Fluorescent images of cells were captured using a LSM 710 laser scanning confocal microscope (Carl Zeiss Microscopy, LLC Thornwood NY).

The mice were transcardially perfused with 0.9% saline (w/v), followed by perfusion with fresh fixative composed of 4% paraformaldehyde in a phosphate buffer (PB, 0.1 M, pH 7.4). The brains were collected and post-fixed overnight before coronal sections (50 μm thickness) were prepared using a vibratome (5100 mz Campden Instruments, Leicestershire, UK). After washing in PB several times, the sections were pre-incubated with 0.3% Triton X-100 (Sigma-Aldrich) in PB for 30 min at room temperature (RT) and incubated overnight with rabbit anti-GFAP antibody (1:1000 dilution, ab7260, Abcam, Cambridge, UK) or mouse anti-HA antibody (1:1000; MMS-101R, BioLegend, San Diego, CA, USA) at RT. Immunofluorescence was performed with secondary antibodies (Alexa Fluor 488-labeled anti-mouse antibody, 1:500; A11001, Invitrogen, Carlsbad, CA, USA or Alexa Fluor 594-labeled anti-rabbit antibody, 1:500; A21209, Invitrogen) for 2 h at RT. The sections were mounted onto glass slides and covered with coverslips using a drop of mounting medium (Dako North America, Carpinteria, CA, USA). The coverslips were sealed with nail polish to prevent the desiccation and movement of the samples under the microscope. Images were obtained using fluorescence microscopy (Axioplan2 Imaging, Carl Zeiss Microimaging, Oberkochen, Germany) and then subjected to analyses.