Optical te2000 s inverted microscope

For the invasion assay, a Transwell chamber coated with Matrigel was used. Following transfection, cells (1.0×10⁵ cells/chamber) were transferred to the upper chamber for incubation while in the lower chamber, DMEM with 20% FBS was added at 37°C. Following 24 h, cells that did not pass through the membrane between the upper and lower chambers were removed using a swab, and those in the lower chamber were fixed with 100% methanol at 20°C for 15 min and then stained with 1% crystal violet at 20°C for 30 min. Cells that passed through the membrane were counted at a magnification of ×20 by the Nikon Optical TE2000-S inverted microscope in six microscopic fields that were selected randomly.

Tissue samples collected in the proliferative and secretory phases were fixed in 10% formaldehyde at 20°C for 24 h, embedded in paraffin and then sliced into sections of 5-µm thickness. The sections were dehydrated in ethanol in a graded concentration series, immersed in xylene at 100°C and alcohol, and then incubated with 1% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 20°C for 1 h and in hydrogen peroxide at 20°C for 30 min. Thereafter, they were probed with primary antibodies against TGF-β (cat. no. 2519; 1:500; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight, followed by three washes in TBS, and they were then probed with horseradish peroxidase-conjugated secondary antibodies (cat. no. 3900; 1:1,000; Cell Signaling Technology, Inc.) for 1 h at 20°C. The sections were then treated with 3,3′-diaminobenzidine at 20°C for 30 min and counterstained with hematoxylin at 20°C for 5 min. Immunostaining was visualized at a magnification of ×40 by the Nikon Optical TE2000-S inverted microscope (Nikon Corporation, Tokyo, Japan).

To quantify the TH immunoreactivity neuronal processes, primary mesencephalic neurons were fixed and followed by immunocytochemistry as described above. Thirty TH immunoreactivity (TH-ir) neurons were randomly selected and captured by a Nikon Optical TE2000-S inverted microscope (Nikon, Melville, New York). The length of each TH-ir cell neurite was traced from the perinuclear region to the end of the neurite using the measurement function of Image J (NIH software). The values were normalized to that obtained from control culture.