Ultraview vox spinning disc confocal microscope

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and sealed with normal goat serum for 30 minutes. After washing for three times in PBST (PBS containing 1‰ Triton X-100), these cells were incubated overnight with the primary antibody at 4°C, and incubated with Alexa Fluor 488 (green) and 546 (red) dye conjugated with Moleculara Probes. The DNA dye DAPI (molecular probe, blue) was used. The confocal scanning analysis was performed with a Ultraview Vox Spinning disc confocal microscope (USA, Perkin Elmer) , in order to minimize the possibility of leakage of fluorescence emission.

FISH was performed in the paraffin sections of tumor nodules from in vivo xenograft assays using a commercial kit from Servicebio (Wuhan, China) according to the manufacturer's protocol. Sections were blocked by goat serum at 37°C for 1 h, and incubated at 4°C overnight with the primary cDNA oligonucleotide probes targeting either FN1 3'-UTR or let-7i-5p. Then, the sections were incubated with secondary antibody conjugated with green or red dye for 1 h. The nucleus was stained using DAPI (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Samples were observed using an Ultraview Vox Spinning Disc confocal microscope (PerkinElmer, MA, USA).
The sequences of the probe targeting FN1 3'-UTR were as follows: 1 (link) 5′-AGACATGCTTGTTCCTCTGGATTGG-3′; 2 (link) 5′-AAGCTGGGTCTGCTAACATCACTCC-3′; 3 (link) 5′-GAGCAAAGGGCTTAAGAAAGAAAGAAG-3′; 4 (link) 5′-GTTGAGCTGAAGCTGGAGAACTTCC-3′; 5 (link) 5′-CAGCCCTCATTTATGAGAAAACCCTC-3′. The sequence of the probe targeting let-7i-5p was 5′-AACAGCACAAACTACTACCTCA-3′.

The cells (5 × 10⁴) were seeded onto glass coverslips in six-well plates and incubated for 24 h. Cells were fixed in 4% paraformaldehyde and blocked with bovine serum albumin. The cells were incubated overnight at 4°C with primary antibodies against E-cadherin (1:200) and N-cadherin (1:100), washed with PBS, and incubated with secondary antibodies (Cy3 conjugated donkey anti-mouse IgG, 1:200, Servicebio, Wuhan, China; FITC-conjugated goat anti-mouse IgG, 1:100, Servicebio, Wuhan, China). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA). Confocal scanning was performed using an Ultraview Vox Spinning Disc confocal microscope (PerkinElmer, MA, USA). The fluorescence intensity of each primary color channel was measured using ImageJ software and the data was illustrated as the mean fluorescence intensity (MFI). A Student's t-test was used to compare the MFIs of different indexes.

Immunofluorescence was performed as previously described [8 (link)]. Briefly, cells were fixed in 4% paraformaldehyde/phosphate buffer solution (PBS) followed by extraction in 0.2% Triton X-100/PBS, or cells were fixed in cold methanol. The cells were sequentially immunostained with the indicated primary and secondary antibodies. DNA was stained with DAPI, and the coverslips were mounted with Mowiol (Sigma). For live-cell imaging of cells co-transfected with GFP-Dzip1 and RFP-Rab8, the original images were collected at 2-μm thickness for five layers every 20 min. For image acquisition and processing, Zen 2009 Light Edition and Velocity (6.1.1) were respectively used for management of the confocal immunofluorescence microscope (Carl Zeiss, LSM-710NLO and DuoScan), and the UltraView Vox spinning disc confocal microscope (PerkinElmer). Super-resolution microscopy was performed with a 3-D structured illumination microscope (Nikon, N-SIM), equipped with 405-, 488-, and 594-nm lasers, electron-multiplying CCD cameras (iXon DU-897E, 512 × 512, 16 μm × 16 μm), and a CFI SR Apo TRIF 100× (1.49NA) oil objective (Nikon). The 3-D images were then reconstructed using the NIS-Elements AR software package (Nikon).

All images and analyses were generated by personnel who had no knowledge of the mouse genotype. Aβ images were obtained using a Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss Microscopy, Jena, Germany) with a 10× lens objective. Mosaic images of the entire cortex and hippocampus of each animal were obtained and analyzed, and plaque burden was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). This was done by isolating the cortex or hippocampus, thresholding to a standard value and calculating the area occupied. Using an UltraVIEW VoX spinning disc confocal microscope (PerkinElmer, Waltham, MA, USA), hippocampal PSD-95 immunoreactive puncta were imaged with a 60× lens objective and digitally magnified to × 100. Two images were obtained in the molecular layer of the dentate gyrus with two slices from each mouse analyzed. Puncta from the dentate gyrus were analyzed and counted using ImageJ, excluding cell somata. Hippocampal calbindin D28 images were obtained using a Zeiss Axio Imager Z1 fluorescence microscope with a 20× lens objective. Mosaic images of the entire hippocampus of each animal were obtained and analyzed. All histologic analyses were done using ImageJ and analyzed statistically by Student’s t-test, or by analysis of variance (ANOVA) with post hoc comparisons as indicated, using SPSS software (IBM, Armonk, NY, USA).