Eclipse te2000 u microscope

For cell proliferation assay, the equal cell number seeded in the 6-well culture plates were transfected with control siRNA or siRNA for MAP4 knockdown. Cell numbers were counted 96-hrs post-siRNA transfection after detaching the cells from the culture plates. For wound healing, cells were seeded to 6-well plates and transfected by control and MAP4 siRNA for 48 hrs until achieving confluence. Then the cells were starved in serum-free medium for 24 hrs and treated with 10 ng/ml EGF. The cellular layer in each plate was scratched using a plastic pipette tip. The migration of the cells at the edge of the scratch was imaged at 0, 6, 12, 24 and 48 hrs using the Nikon Eclipse TE2000-U microscope (Nikon Instruments Inc) and quantified by ImageJ.
The bottom polycarbonate filter surface of Transwell inserts (8 μm pores; Corning) was coated with 10 μg/ml of LN332 (Kerafast) diluted in PBS for 3 hrs at 37^oC. Cells (5×10⁴) 48 hrs post-transfection of control or MAP4 siRNA were suspended in serum-free medium containing 1% BSA and then were plated in the upper insert chamber with or without 10 ng/ml EGF. Cells were allowed to migrate/invade for 16 hrs at 37°C. Cells on the bottom of the filter were then fixed with 4% PFA and stained with 0.1% Crystal Violet. Then the cells were imaged by the Nikon Eclipse TE2000U microscope and quantified by ImageJ.

Standard procedures were used for detection of Nissl bodies found within neurons as has been previously described (Talley Watts et al., 2013 (link)). Briefly, brains were harvested, sectioned at 20um and placed on plus slides. The slides were dried at 37°C overnight. Slides were hydrated with distilled water, 0.1% cresyl violet was applied for 7 minutes, followed by a wash in distilled water. The slides were then hydrated, cleared in xylene and cover slipped. Images were acquired on a Nikon Eclipse TE2000-U microscope (Nikon Inc.). Cell area was quantified using the ImageJ analyze particle function. This function allows for the exclusion of particles below 100 pixels, resulting in the collection of data from only those particles from cell somas. Soma size was determined in the ipsilateral cortex of 5 cryosections taken from the area of impact. The spinal cord sections were taken from the lumbar spinal cord 24 hours post injury or sham surgery and the soma size was determined in the contralateral lumbar spinal cord to assess the changes in the motor track associated with the ipsilateral brain trauma.