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7 protocols using plan neofluar 10x 0

1

Detailed Amygdalar Parcellation Technique

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A low power stereomicroscope was used to examine the sections and camera lucida drawings outlining architectural borders were made. Architectonic borders of the amygdaloid nuclei and cortical regions were first defined using the standard Nissl and myelin stains. The parcellation of the nuclear and cortical regions of the amygdaloid body was then confirmed and refined using the immunohistochemical stains. The drawings were then scanned and redrawn using the Canvas Draw 6 drawing program (Canvas GFX, Inc., FL, USA). The nomenclature used in the current study was based primarily on that used by Paxinos et al. (2009 ), Price et al. (1987 ), Sah et al. (2003 (link)), Radtke‐Schuller (2018 ), and Pillay et al. (2021 (link)). While amygdaloid terminology varies across studies and species (e.g., Sorvari, Soininen et al., 1995 (link); Pillay et al., 2021 (link)), where the terminology used in these studies was not appropriate to the current findings, we used the most appropriate terminology available. Digital photomicrographs were captured using an Axiocam 208 color camera mounted to a Zeiss Axioskop microscope (with Zeiss A‐Plan 5X/012, Zeiss Plan‐NeoFluar 10X/0.30, and Zeiss Plan‐NeoFluar 40X/0.75 objectives). No pixilation adjustments, or manipulation of the captured images were undertaken, except for the adjustment of contrast, brightness, and levels using Adobe Photoshop.
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2

Cortical Mapping of Neuronal Markers

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Images were acquired on a Zeiss LSM 710 confocal microscope. The cortical distribution of WFA-FITC, Agg and PV immunoreactivity was examined in a z-stack (3 × 10 μm images) acquired with a 10X lens (Zeiss Plan-neofluar 10x/0.30, NA = 0.30) and a z-stack (9–11 × 4.5 μm images) acquired with a 100X (Zeiss Plan-neofluar 100x/1.4 Oil DIC, NA = 1.3). Maximal intensity projections (MIPs; 450 μm width, 0–750 μm from cortical surface) were used to obtain mean intensity profiles in Fiji (NIH). Co-localization of MMP2/9 biomarker puncta with VGluT2 was analyzed in a single Z-section image taken at 40X, using Fiji. After the threshold function (auto threshold + 25) was applied to MMP2/9 biomarker and VGluT2 puncta, co-localized puncta were identified by size exclusion (0.2 μm2 < 2.0 μm2) using the ‘analyze particles’ function in Fiji. PV+ somata were identified by size exclusion (20–200 μm2) and fluorescence intensity (auto threshold + 25). Co-localization with VGluT2 was re-quantified following 2 μm shift of MMP2/9 biomarker images.
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3

Confocal Imaging of GapYFP in Cells

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Images were collected on an inverted laser scanning confocal microscope (LSM5 Pascal, Carl Zeiss, Thornwood, NY) using either a Plan-Neofluar 10X/0.3, Plan-Neofluar 40X/0.75 or C-Apochromat 40X/1.2W objective (Carl Zeiss, Thornwood, NY). The GapYFP was excited with the 488 nm laser line using the FITC filter and all other imaging parameters were as described in (36 ). Images were collected, processed and analyzed using AIM software (Carl Zeiss, Thornwood, NY).
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4

Time-lapse Confocal Imaging of Embryo Explants

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3D image z-stacks were collected on an inverted laser scanning confocal microscope (LSM5 Pascal, Carl Zeiss) using either a Plan-Neofluar 10X/0.3, Plan-Neofluar 40X/0.75 or C-Apochromat 40X/1.2W objective (Carl Zeiss). For embryo explant time-lapse microscopy, the microscope was surrounded with a snug fitting cardboard box and thermal insulation (Reflectix, BP24025, Markelville, IN) with a table top incubator (Lyon Electric, 950-107, Chula Vista, CA) fed into one side of the box (Kulesa and Kasemeier-Kulesa, 2007). The EGFP plasmid was excited with the 488 nm laser line using the FITC filter. Time-lapse images were recorded every 5 minutes for an average of 12–16 hours. Images were collected, processed and analyzed using AIM software (Carl Zeiss) and ImageJ v1.30 software (developed at NIH and available on the Internet at http://rsb.info.nih.gov/ij/). Statistical analysis was performed using the Student’s t-test.
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5

Imaging Techniques with Zeiss Confocal

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Images were acquired using a LSM810 confocal microscope (Zeiss) equipped with an EC Plan-Neofluar 10x/0.3, a Plan-Apochromat 20x/0.8, a Plan-Apochromat 40x/1.4 Oil DIC, or a Plan-Apochromat 63x/1.40 Oil DIC objective. Images were taken using Zen2.1 software (Zeiss), and processed and quantified with Fiji (NIH). Figures were assembled using Adobe Photoshop and Illustrator.
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6

Confocal Microscopy Imaging Workflow

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Images were acquired using a LSM810 confocal microscope (Zeiss) equipped with an EC Plan-Neofluar 10 x/0.3, a Plan-Apochromat 20 x/0.8 or a Plan-Apochromat 40 x/1.4 Oil DIC objective. Images were taken using Zen2.1 software (Zeiss) and processed and quantified with Fiji (NIH). Figures were assembled using Affinity Photo and Windows Office Powerpoint.
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7

Confocal Microscopy Imaging Workflow

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Images were acquired using a LSM810 confocal microscope (Zeiss) equipped with an EC Plan-Neofluar 10x/0.3, a Plan-Apochromat 20x/0.8 or a Plan-Apochromat 40x/1.4 Oil DIC objective.
Images were taken using Zen2.1 software (Zeiss) and processed and quantified with Fiji (NIH). Figures were assembled using Adobe Affinity Photo and Windows Office Powerpoint.
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