Double-IF images of Iba1+ microglia and Bassoon+ synaptic particles were taken using sequential acquisition of separate wavelength channels by confocal laser scanning microscopy (Spinning disk, Visitron CSU-W1, Visitron Systems, Puchheim, Germany) with a 100× (oil, NA 1.4) objective. Laser intensities for each channel were set and kept constant during the entire image acquisition. For each animal, 12 series of images with a minimal z-stack distance of 0.13 μm were randomly acquired from three consecutive sections containing the medial portion of the PFC spanning the IL, PL, and AC subregions (bregma: +2.2 to +1.4 mm). All images were deconvolved with Huygens Professional version 20.10 (Scientific Volume Imaging, The Netherlands, http://svi.nl ) using the CMLE algorithm with SNR:10 and 40 iterations. After deconvolution, the images were imported into Imaris image analysis software (version 9.6.0, Oxford Instruments). Iba1+ microglia were reconstructed three-dimensionally using the “surface creation” wizard. Bassoon+ synaptic puncta were generated using the “spots creation” wizard with default parameters. The “split into surface objects” function was used to quantify Bassoon+ synaptic particles within surface-rendered Iba1+ microglia. The colocalizing synaptic particles were normalized to the volume of microglia.
Imaris image analysis software
Manufactured by Oxford Instruments
Sourced in United Kingdom, Switzerland
Imaris is a 3D and 4D image analysis software developed by Oxford Instruments. It provides advanced image processing and visualization capabilities for a wide range of scientific applications.
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Lab products found in correlation
Tcs sp8, by Leica (2 mentions)
Sp8 lightning confocal microscope, by Leica (1 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Tween 20, by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Citrisolv, by Thermo Fisher Scientific (1 mentions)
Anti phospho histone h3, by Cell Signaling Technology (1 mentions)
W32466, by Thermo Fisher Scientific (1 mentions)
A34055, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Stellaris 8 confocal microscope, by Leica (1 mentions)
Cytospin 4, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
Calcein am, by Abcam (1 mentions)
Draq5, by Abcam (1 mentions)
Lsm 980 confocal microscope, by Zeiss (1 mentions)
19 protocols using imaris image analysis software
1
Quantifying Microglial Synaptic Engulfment
2
Visualizing Dendritic SAP97 Localization
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DG granule neurons in organotypic entorhino-hippocampal slice cultures made from P6-P8 rats were biolistically co-transfected with mCherry-tagged βSAP97 constructs (wild-type, G344R, or G357S) and the GFP expressing pFUGW-βSAP97-miR construct ~18-20 h after plating. Slices were fixed in 4% PFA/4% Sucrose in PBS and washed 3x with PBS. Slices were further processed with an abbreviated SeeDB-based protocol for imaging54 (link),84 (link),85 (link). Images were acquired at DIV7 via multiphoton confocal microscopy (SP8 LIGHTNING Confocal Microscope, Leica). Images were acquired using a 63x/1.4NA oil immersion objective. Imaris image analysis software (Oxford Instruments) was used to identify and visualize dendritic regions exhibiting the highest βSAP97 fluorescent intensity.
3
Sholl Analysis of Prefrontal Cortex Neurons
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The complexity of dendritic arbors of biocytin-labeled layer 2/3 pyramidal neurons of the PFC was assessed by means of the Sholl analysis (60 (link)). Images were captured with confocal laser scanning microscopy (Leica DMI6000 AFC, Model SP8, Mannheim, Germany) using a 20× (oil, NA 0.75) objective with a minimal z-stack distance of 2 μm. All images were deconvolved with Huygens Professional version 20.10 (Scientific Volume Imaging, The Netherlands, http://svi.nl ), using the CMLE algorithm, with SNR:10 and 40 iterations. After deconvolution, the images were imported into Imaris image analysis software (version 9.6.0, Oxford Instruments). Neurons were reconstructed three-dimensionally using the “filament creation” wizard. Manual error correction was performed to ensure accurate dendrite rendering. For the Sholl analysis, a sphere radius gap of 20 μm was chosen, and the number of dendrite intersections against the radial distance from soma was quantified automatically by the software. On average, five to six pyramidal neurons per animal were included in the Sholl analysis. To avoid spurious findings arising from pseudoreplication, the number of animals (N = 7 to 8 per treatment) was considered as the experimental unit in statistical analyses (see below).
4
Time-lapse Imaging of Fluorescent Cells
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Fluorescently labeled cells were plated onto sterilized four-chamber 0.170-mm glass-bottom slides (80427, Ibidi) at a density of 1 × 104 cells per well. The following day, cells were imaged with an Andor Revolution XDi WD Spinning Disk Confocal microscope on a humidified stage, which was kept at 37°C with 5% CO2 to simulate optimal growing conditions. Hepes (10 mM) (25-060-Cl; Corning) buffer was added to the culture medium before imaging to maintain a stable pH throughout imaging. Cells were imaged every 5 min for movie S1 and every 10 min for movie S2 using a 63× silicon immersion objective lens for 12 hours. Images and movies were analyzed with Imaris Image Analysis Software (Bitplane, Oxford Instruments). For labeling of cell membranes, CellMask Deep Red (C10046, Thermo Fisher Scientific) was used according to the manufacturer’s protocol.
5
Immunofluorescence Analysis of Mitosis and Apoptosis
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Samples were fixed overnight at 4°C in 4% paraformaldehyde, then dehydrated and infiltrated with paraffin by hand. After sectioning, samples were dewaxed, subjected to antigen retrieval and stained according to Anderson et al. (2016a) (link) with the following modifications: Citrisolv (Thermo Fisher Scientific) was used for dewaxing, sodium citrate buffer containing 0.05% Tween 20 (Sigma-Aldrich) and 10% normal goat serum (Sigma-Aldrich) in PTX was used for dilution of blocking and antibodies. Primary antibodies used were anti-phospho histone H3 (Cell Signaling Technologies, 9706L, 1:500) and anti-cleaved caspase 3 (Cell Signaling Technologies, 9661L, 1:250). Secondary antibodies used were goat anti-mouse Alexa Fluor 594 (Thermo Fisher Scientific, A-32740) and goat anti-rabbit Alexa Fluor 647 (Thermo Fisher Scientific, A-21244). Immunocomplexes were detected using a Zeiss LSM710 confocal microscope using a 20× objective to generate tiled z-stacks. Nuclei were counted using Imaris image analysis software (Oxford instruments). pHH3- and cleaved caspase 3-positive cells were counted manually using maximum projections of z-stacks. Three sections per embryo were analyzed to generate results in triplicate.
6
Confocal Microscopy of Pupal Wing Tissue
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For confocal microscopy of fixed tissue, pupal wings were dissected and fixed in PEM buffer (0.1 mol l−1 PIPES, 2 mmol l−1 EGTA, 1 mmol l−1 MgSO4, pH 6.95) with 3.7% paraformaldehyde for 20–30 min at room temperature, as described previously (Dinwiddie et al., 2014) (link). Fixed wings were incubated in 1X PBS+0.1% Triton-X 100 (PT) with 1:200 dilution of phalloidin, Alexa 555 conjugated (Invitrogen A34055), and wheat germ agglutinin, Alexa 647 conjugated (Invitrogen W32466) at a dilution of 1:200 overnight at 4°C. Wings were washed in PT and then placed in 50% glycerol:PBS with 1 µg ml−1 DAPI overnight at 4°C. Wing samples were placed on microscope slides and mounted in 70% glycerol:PBS. A coverslip (#1.5 thickness) was applied, and each preparation was sealed around the edges with nail polish. Slides of fixed tissue were examined with an LSM 880 confocal microscope (Carl Zeiss, Germany) with 40× and 63× objectives. Confocal images and movies were generated using Imaris Image Analysis Software (Bitplane, Oxford Instruments, UK).
7
Immunofluorescence Staining of Organoid Cultures
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24-well Dome cultures were fixed with 2% paraformaldehyde (PFA) in PBS. Domes were detached from plate using a spatula, and transferred into microcentrifuge tubes using a cut P1000 pipette tip. For BOBA cultures, 500 uL of culture was transferred to a microcentrifuge tube using a cut P1000 pipette tip, the media was removed and 2% PFA in PBS was added. Samples were incubated in fixative at RT for 15–30 min, then washed 3X in PBS. Samples were stained in microcentrifuge tubes with primary antibodies diluted in Blocking/Permeabilization Buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS) for at least 4 h at RT, then washed 3X in PBS. Primary antibodies used are as follows: ɑ-Ki67 (Invitrogen cat. no. MA5-14520), ɑ-MUC2 (Millipore cat. no. MABF1989), ɑ-FABP1 (Novus cat. no. NBP-87695), and ɑ-CHGA (Novus cat. no. NB120-15160). Samples were then incubated with secondary antibodies (donkey ɑ-rabbit Alexa Fluor 488 (ThermoFisher cat. no. A-21206) or goat ɑ-mouse Alexa Fluor 594 (ThermoFisher cat. no. A-11032)), with DAPI, and AlexaFluor 660 Phalloidin diluted in Blocking/Permeabilization Buffer at RT for at least 2 h at RT. Images were collected on a Stellaris 8 Confocal Microscope (Leica) using a 40X objective and 3D-reconstructed using Imaris Image Analysis software (Oxford Instruments).
8
Quantifying α Cell Proliferation in Slc7a2 Knockout Mice
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Ex vivo α cell proliferation was assessed as described previously (10 (link)). Briefly, islets isolated from Slc7a2−/−, Slc7a2+/− and Slc7a2+/+ mice were cultured in DMEM-based medium with high or low amino acid concentrations, based on amino acid levels in Gcgr−/− and Gcgr+/+ mouse serum, respectively (See Supplemental Table 1 for amino acid concentrations in islet culture media), for 72 hours. After culture, islets were washed in 2mM EDTA and dispersed with 0.025% Trypsin at 37°C for 10 minutes with mixing. Dispersed islet cells were recovered by centrifugation in RPMI media containing 5.6mM glucose, 10% FBS, and 1% Penicillin/Streptomycin. The resulting cell pellet was resuspended in medium and centrifuged onto a glass slide using a Cytospin 4 (Thermo Scientific, Waltham, MA) centrifuge. Slides were air-dried for 30 minutes, then stored at −80C until use. For staining, slides were thawed, immediately fixed in 4% paraformaldehyde and immunostained for glucagon, to mark α cells, and Ki67, to mark proliferating cells. Slides were imaged using a Leica Microsystems Epifluorescent Microscope DM1 6000B. The percent of proliferating α cells was calculated based on the number of Ki67+/glucagon+ cells divided by the total number of glucagon+ cells using Imaris image analysis software (Oxford Instruments).
9
3D Quantification of mGRASP-Labeled Synapses
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To analyze synapses labeled by mGRASP, ACC‐containing sections were imaged using a confocal microscope FV3000 (Olympus, Japan). Multiple optical sections (0.5 mm thickness) were imaged to cover the entire z‐axis of the section. Thereafter images were reconstructed in 3D and analyzed using Imaris Image analysis software (Imaris 9.0.0, Oxford Instruments). 3D isosurfaces were created for each PV+, SST+, or CaMKII+ neuron identified by the tdTomato signal expressed in post‐mGRASP neurons and were masked to isolate the fluorescent signals within and surrounding the cell body. For each masked cell, a second round of 3D isosurfaces was created for the mGRASP signal to form a mask around all mGRASP labeled synapses. To avoid false‐positive counting of synapses, it was visually confirmed that the synapses were formed on postsynaptic cortical neurons with thalamic axons identified with the pre mGRASP mCerulean signal. Care was taken to ensure that the entire mGRASP signal was covered by the isosurfaces created. The number of such isosurfaces created was used to quantify the number of synapses per cell, while their volumes were used to quantify the volume of the synapses.
10
Human iPSC-derived NPC Viability Assay
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Human iPSC-derived NPCs were encapsulated within bulk and granular hydrogels, as well as seeded onto control tissue culture polystyrene (TCP) plates in Neural Basal Medium supplemented with 10 ng/ml BDNF and 10 ng/ml GDNF. The cell seeding densities were ~107 cells/ml and 5 × 104/cm2 for the hydrogels and TCP controls, respectively. Cell viability was evaluated on Day 1, Day 3, and Day 7 using a live cell stain, Calcein AM (Abcam), and DRAQ5™ (Abcam), which is a cell permeable far-red fluorescent DNA dye staining cell nuclei. Images for cell viability assay were acquired with a Nikon TiE C2+ confocal microscope (Nikon Instruments Inc., UK) by sequential scanning. The thickness of the acquired hydrogel sections was about 500 μm and z stacks of typically 79 × 6.5 μm slices were imaged. The cell viability was determined by the percentage of the number of viable cells to the total number of cell nuclei analyzed using Imaris Image Analysis Software (Oxford Instruments plc, UK). A filter was applied to remove the background noise based on the particle size (>524 μm3), which correlates to the soma diameter of the NPCs of 10 μm.
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