Csu x1 spinning disc unit

CHO cells were analyzed by fluorescence microscopy using an Olympus Confocal FV1000. The program FV10-ASW 3.0 was used for all acquisitions and settings for the live imaging. For spinning disk acquisition, we used a Quorum Technologies WaveFX Confocal Microscope with a Yokogawa CSU-X1 Spinning Disc Unit and a LCI Temperature Controller and CO₂ Mixer for Live Cell Imaging (Neurobiology of Stress CNS, University of Toronto at Scarborough, Canada). The resulting movies series were corrected to improve the contrast and resolution using the deconvolution plugin on ImageJ. The image analysis of Saf growth and contraction was carried out using ImageJ.

To perform immunostaining of biocytin filled neurons, slices containing biocytin filled neurons were fixed in 4% PFA overnight at 4°C. After fixation, slices were transferred to PBS. Biocytin filled INs (n = 8 Sst⁺ Group 1; 3 Sst⁺ Group 2; 6 PV⁺) were blocked (1X PBS, 0.3% triton, 5% BSA, 5% Normal Donkey Serum) for 4 hours before 24-hour incubation with streptavidin conjugated Alexa Fluor 488 (1:500, ThermoFisher Scientific, Waltham, MA, USA) at 4°C. Slices were mounted with Prolong Gold and sealed for long-term storage. Slices were imaged using an Axio Observer microscope (Carl Zeiss, Okerkochen, Germany); equipped with a CSU-X1 spinning disc unit (Yokogawa, Musashino, Tokyo, Japan); 488 nm/40 mW laser; Plan-NeoFluar 40X (0.75 NA) air objective lens; and Evolve 512 EM-CCD camera (Photometrics, Tucson, AZ, USA). SlideBook 6.0 software (3i, Denver, CO, USA) enabled instrument control and data acquisition. Images were acquired in sections by following branched points from the cell soma. Images were stitched in Fiji software using Grid/Collection stitching (Preibisch et al., 2009 (link)) with an unknown position type.

Fluorescence microscopy was performed with a Zeiss Observer SD spinning disk confocal microscope using a Yokogawa CSU-X1 spinning disc unit and an oil objective (63× magnification) and BP 525/50 (WGA488) and LP 690/50 (Atto633) emission filters. A 488 nm and a 639 nm laser were used for excitation. At 24 h prior to transfection, 3500 T24 cells per well were seeded in 8-well plates in 280 µL growth medium. A total of 20 µL MSN-454-GE11 covalently labeled with Atto-633 carboxy (200 µg mL⁻¹ MSN in HEPES buffer) were added to each well and incubated for 45 min or 6 h, respectively, at 37 °C followed by addition of WGA-488 to the medium for cell membrane staining. Cells were washed with PBS and after addition of 300 µL fresh medium, cells were directly imaged.
The cellular uptake of nanoparticles was quantified using the ImageJ macro “Particle_in_Cell-3D” developed by Adriano A. Torrano and Julia Blechinger, Department of Chemistry and Center for NanoScience (CeNS, University of Munich (LMU), Munich, Germany. http://imagejdocu.tudor.lu/doku.php?id=macro:particle_in_cell-3d)

Images were acquired on an Olympus 1X81 fluorescence microscope equipped with a CSU-X1 spinning-disc unit (YOKOGAWA) or a Nikon-A1 laser scanning confocal microscope (Nikon). Fluorescence images acquired from confocal microscopy were reconstructed in 3D using Imaris8.2 (Bitplane). Measurements of cell nucleus dimensions, cyst epithelial thickness, and cyst orientation angle were performed manually using the Measurement tool in ImageJ (NIH)^{39 (link)}. When necessary, 3D reconstructed cyst images were rotated to render a cross-section appropriate for measurements. When conducting immunofluorescence analysis on PASE, we have cross-checked HOECHST staining with other antibody staining to ensure that (1) the amniotic ectoderm contains cells that are double positive for both HOECHST and corresponding amniotic marker such as TFAP2A and GATA3; (2) the amniotic ectoderm contains HOECHST positive nuclei that are negative for pluripotency markers such as NANOG and OCT4.

The fluorescence
microscope images
were taken with a Zeiss Observer SD spinning disk confocal microscope
using a Yokogawa CSU-X1 spinning disc unit and an oil objective with
63× or 100× magnification (1.40 N.A.) and BP 525/50 (green
channel) and LP 690/50 filters (red channel). For excitation, a 488
nm and a 639 nm laser were used. The images were processed with the
ImageJ v2.35 software.