Lsm 510 carl confocal microscope

The L2-L1 or L5-L6 segments of spinal cords were dissected and fixed in 4% paraformaldehyde for at least 4 hr at room temperature. They were then transferred to phosphate buffered saline (PBS), and embedded in a 5% Agar/PBS solution. Transverse sections (60 μm) were cut on V1000 vibratome (Leica Biosystems, Buffalo Grove, IL). The sections were blocked in 10% normal donkey serum in 0.01M PBS with 0.1% Triton X-100 for 90 min, and subsequently incubated overnight at room temperature in Chicken Anti-GPF antibody (ab13970, dilution 1:1000, Abcam, Cambridge, MA), Goat Anti-ChAT antibody (AB144P, dilution 1:100, Millipore, Temecula, CA). The following day, the sections were washed for an hour and then incubated for 3 hr in secondary antibodies: Donkey Anti-Chicken-FITC and Donkey Anti-Goat-Cy5 (dilution 1:50, Jackson ImmunoResarch, West Grove, PA). The sections were then mounted on slides and cover-slipped with a Glycerol/PBS solution (3:7). Images were acquired using a LSM510 Carl Zeiss confocal microscope with either 10X (air) and 40X (oil) objectives.

Untransfected, HeLa cells expressing different forms of gB or gH were grown to 80–90 % confluence in a 1 % collagen coated 24-well plates. The monolayers of cells were wounded by performing a scratch with a sterile 1000 μl micropipette tip as described earlier [22 (link)]. Cells were washed with DMEM, and further incubated at 37 °C in DMEM containing 2 % FBS. Wound closure was monitored at 16 h post scratch and imaged with a laser-scanning LSM 510 Carl Zeiss confocal microscope (Magnification, x 20 objective). The open area (scratch) was quantified with TScratch software [23 (link)].