90i upright fluorescence microscope

8μm normal oral gingival mucosa and OSCC sections were immunofluorescence stained according to our established protocols.^{16 –19} Sections were mounted with DAPI contained Vectashield mounting medium (Vector Laboratories), and imaged with the Nikon A1 confocal system (Nikon Instruments Inc., Melville, NY) on a Nikon 90i upright fluorescence microscope.
2.5×10⁴ UMSCC 38 and 11B cells/well were cultured in 10% FBS/DMEM on glass coverslips until they reached 40~60% confluence. Cells were washed with sterilized PBS twice prior to being applied to a 48 h serum-free starvation (the synchronization) in DMEM, and then treated with TGFβ1 in 0.2% FBS DMEM. The subsequent fixation and staining of cells were carried out as previously described by us.^{16 –19} Stained cells were mounted with Vectashield mounting medium containing DAPI. All images were obtained on an inverted motorized microscope (Axiovert 200M, Zeiss) using SlideBook 5.0 image acquisition software (Intelligent Imaging Innovations Inc, Denver). Five microscopic fields were selected randomly (40× magnificent), in which, both immunofluorescence and DAPI stained cells were counted manually as the number of positive stained cells and total cells respectively; the ratio of the positively stained cells was then calculated.

Stereological analysis of dopamine neuron number in the SN was achieved using an adapted protocol from Tapias et al. (2013), employing an unbiased, automated system. Briefly, nigral tissue sections were stained for TH and counterstained with DAPI and NeuroTrace Dye (640; Life Technologies) and imaged using a Nikon 90i upright fluorescence microscope equipped with high N.A. plan fluor/apochromat objectives, Renishaw linear encoded microscope stage (Prior Electronics) and Q-imaging Retiga cooled CCD camera (Center for Biological Imaging, University of Pittsburgh). Images were processed using Nikon NIS-Elements software, and quantitative analysis was performed on fluorescent images colocalizing DAPI, TH, and Nissl-positive stains.