Leica microscope imaging software

Sections were rinsed for 6 × 5 minutes in phosphate-buffered saline (PBS) and incubated for 1 hour in a blocking solution comprising of PBS with 0.3% (w) Triton X-100 and 5% (w) donkey serum (Abcam) containing 1% (w/v) bovine serum (Sigma). They were then incubated for 15 hours at 4 °C in blocking solution containing chicken anti-GFP (1:1000, AB13970, Abcam) and goat anti-ChAT (1:500, AB144, Milipore) antibodies. The sections were then rinsed for 6 × 5 minutes in (PBS), then incubated for 2 hours in blocking solution containing goat anti-chicken Alexa Fluor 488 (1:1000, 11039, Life technologies) and donkey anti-goat Alexa Fluor 594 (1:1000, AB150132, Abcam) at room temperature. After 6 × 5 minutes rinse, the sections were mounted in Fluoroshield with DAPI (Sigma). Fluorescence images to verify expression of the eYFP/GFP tag and to visualise ChAT labelled neurons were taken with a Leica microsystems SP8 confocal microscope using a 10× and 20× lens and acquired with Leica Microscope Imaging Software.

SMMC-7721 and Bel-7402 cells were treated with various concentrations of CT-707 in the first 24 h and then with various doses of sorafenib for 48 h in normoxic or hypoxic condition, and cell proliferation were measured by microscopy (Leica DM4000 B) and sulforhodamine B (SRB) protein assay (Sigma, S1402) [14 (link)]. For SRB, Cells (7000/well) were incubated with 10% trichloroacetic acid (TCA) for 1 h (4 °C) and washed it at least 3 times by water, then stained with sulforhodamine B for 30 min. The sulforhodamine B was washed away with 1% cold acetic acid, and 100 μl of 1% Tris-base was added to each well. The optical density (OD) was determined at 515 nm by a Multiskan Spectrum plate reader (Thermo Electron Corporation, Marietta, OH, USA). For clonogenic assay, Cells were observed and photographed by 10X objective, the results were analyzed by Leica Microscope Imaging Software.