The fluorescent in situ assay shown in Figures 5 C, 5E, 6 I, S4 B, S4C, and S7 G–S7I was performed using the RNAscope 2.5 HD—multiplex fluorescent Manual Assay kit (ACDbio Cat. 320850). In this assay tissue was prepared and pretreated in the same way as in the chromogenic assay (see above). After incubation with target probes, 3 amplification steps and 1 detection step were performed following manufacturer instructions. Slides were mounted using the SlowFade Gold with DAPI mounting media (Life Tech Cat. S36939) covered with 1.5 glass coverslip (Fisher Cat. 12544E) and sealed with clear nail polish. Slides were imaged using the Axio Imager.Z2 fluorescent microscope (Zeiss). A 16 bit 1388 X 1040 pixel images were acquired using AxioCam HR3 camera (Zeiss). Fluorescent signal was imaged at 20x magnification using the Apotome.2 module (Zeiss). Z stack images (optical slice 1 μm) were obtained for each image. Set exposure was used when comparing signal between WT and KO mice for a given experiment. For cell type specific expression quantification experiments shown in Figures 5 C–5E slides were imaged using the Zeiss LSM 710 confocal microscope at 20X magnification. 16 bit images at 0.16 μm/pixel were generated. A z stack (optical slice 1 μm, 6 slices total) was obtained for each image. Example images show maximal intensity projections for each group.
Axiocam hr3 camera
Manufactured by Zeiss
Sourced in Germany
The AxioCam HR3 is a high-resolution digital camera designed for microscopy applications. It features a 3.3-megapixel CMOS sensor and is capable of capturing images with a resolution of up to 2048 x 1536 pixels. The camera is equipped with a C-mount interface for attachment to various microscopes.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Axio imager z2 fluorescent microscope, by Zeiss (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Apotome 2 module, by Zeiss (2 mentions)
Slowfade gold with dapi mounting media, by Thermo Fisher Scientific (2 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Axioskop 40, by Zeiss (1 mentions)
7 protocols using axiocam hr3 camera
1
Multiplex Fluorescent In Situ Hybridization
2
Quantifying Bacterial Biomass and Cell Counts
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The bacterial biomass and cells counts were analysed by epifluorescence microscopy (Buesing 2005 ) on a Carl Zeiss Axio Imager Z1 microscope (1000×). Sampling and analysis were performed at day 0, 29, 31, 74 and 77. Digital images were taken randomly (10 fields using an AxioCam HR3 camera) and processed in AxioVision Version 6.1.7601 (Carl Zeiss MicroImaging, Germany). Every cell was manually marked, and the software delivered the spatial dimensions for each marked cell. The cell volumes were calculated as rods or spheres according to the closest geometric shape. The biomass was calculated as the cell carbon content from mean biovolumes and abundance using a relationship proposed by Romanova and Sazhin (2010 ): fg C cell−1 = 133.754V0.438, where V is the cell volume in μm3.
3
Astrocyte Development Imaging Protocol
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Imaging was performed using an Axio Imager.Z2 fluorescent microscope (Zeiss) with the apotome module (apotome 2.0) and AxioCam HR3 camera (Zeiss) at 20× magnification. Tile images that contain the entire primary VC (from pial surface to white matter tract) were acquired. Number of tiles adjusted to contain a similar area of the cortex at each developmental stage, typically 1–2 (width) × 2–4 (depth) (pixel size 0.3 × 0.3 µm).
For RiboTag validation (Figure 1 , Figure 1—figure supplement 1 ): Single-plane images were obtained.
For in situ hybridization experiments (Figure 2 , Figure 2—figure supplement 1 ): z-stack images (seven slices, optical slice 1 µm) were obtained.
For developmental analysis of astrocyte numbers per layer (Figure 2 , Figure 2—figure supplement 1 ), presynaptic marker analysis during development (Figure 3 , Figure 3—figure supplement 1 ), VGlut2 cKO validation (Figure 4 , Figure 4—figure supplement 2 ), and Aldh1l1-GFP mouse validation (Figure 2—figure supplement 1 ): z stack images (three slices, optical slice 1 µm) were obtained.
For RiboTag validation (
For in situ hybridization experiments (
For developmental analysis of astrocyte numbers per layer (
4
Analyzing Cell Adhesion Dynamics
5
Quantitative Ultrastructural Analysis of Peripheral Nerve Axons
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PNVAs of similar length taken from similar regions of the thigh were fixed and embedded similar to previous descriptions (Mikesh et al., 2018; Smith et al., 2020). PNVAs (n = 17) were fixed overnight at room temperature in 2% paraformaldehyde/3% glutaraldehyde fixatives (15713, 16220; Electron Microscopy Sciences [EMS], Hatfield, PA, USA) in 0.1 M sodium cacodylate buffer (11653; EMS). Tissues were washed with buffer prior to trimming and post-fixing in 1% osmium tetroxide/1% potassium ferrocyanide (19150; EMS) in 0.1 M sodium cacodylate buffer for 4–5 hours. PNVAs were then washed in water, stained in 1% aqueous uranyl acetate (22400-1; EMS) for 2 hours, and then washed and held in water. PNVAs were dehydrated through graded ethanol, exchanged to absolute acetone, placed in increasing concentrations of Hard Plus Resin 812 (14115; EMS), and then embedded in fresh resin and polymerized at 60°C for at least 48 hours. Glass knife-thick sections (0.5 mm) were stained in toluidine blue (22050; EMS), and images were captured by Axiovert 200 M microscope (Zeiss, Oberkochen, Germany) using an Axiocam HR3 camera (Zeiss). For each PNVA sample, three regions of interest were given blinded to two investigators, and manually annotated for a total of at least 150 axons. Five different categories of axons were then reported as percentages of the total number of axons.
6
Fluorescent in situ Hybridization Assay
7
Microscopic Observation of LLAI Cells
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An optical microscope (Axioskop 40 equipped with Axiocam HR3 camera, Carl Zeiss Microscopy, LLC, Thornwood, NY) was used for morphological observation of LLAI cells. The objectives and contrast setting used for 400 and 1000× magnifications were A-plan 40×/0.65 with phase-2 contrast and A-plan 100×/1.25 oil with phase-3 contrast, respectively.
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