Fv1000 filter confocal microscope

Spinal cord sections from sham and SCI mice at 3, 14, 21 and 28 dpi were immunostained for the pre-synaptic protein VGlut2 (Figure S1). Stained sections were imaged and analyzed using a modified protocol from Ippolito and Eroglu (2010) (link). Briefly, 15-step Z stacks of 0.33 μm increments spanning 5 μm were acquired on an Olympus Filter FV1000 Confocal microscope with 40x magnification and 2x optical zoom (80x final magnification). Imaging parameters were consistent to minimize intensity variability. Z stack steps were compressed serially in groups of three (i.e., steps 1–3, 4–6, 7–9, 11–12, 13–15) to create 5 maximum intensity projections (MIPs). The ImageJ plug-in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) was used to threshold VGlut2⁺ signal and count the number of labeled puncta. Details of the method can be found in Ippolito and Eroglu (2010) (link).

Each spinal level from T1-T10 was immunostained for the pre- and post-synaptic proteins markers, VGlut2 and Homer1, respectively. Stained sections were imaged and analyzed using a modified protocol from Ippolito and Eroglu (2010) (link). Briefly, 15-step Z stacks of 0.33 μm increments spanning 5 μm were acquired on an Olympus Filter FV1000 Confocal microscope with 40x magnification and 2x optical zoom (80x final magnification). Imaging parameters were consistent to minimize intensity variability. Z stack steps were compressed serially in groups of three (i.e., steps 1–3, 4–6, 7–9, 11–12, 13–15) to create 5 MIPs. Synapse quantification was performed with MIPAR Image Analysis software with a custom semi-automated synapse algorithm (https://www.mipar.us/recipe-store.html). Briefly, the intensity of VGlut2⁺ and Homer1⁺ channels was gated based on the median intensity MIP from each image. Puncta > 100 pixels or < 5 pixels were removed from the channel. Puncta in one channel that met these criteria and overlapped spatially with puncta from the other channel were counted as a functional synapse. For representative images (Figures 1 and 6), MIPs of fluorescent images were imported into Adobe Photoshop and a levels adjustment layer was applied to reveal the full tonal range of visible pixels in the RGB histogram.

All animal experiments were conducted in accordance with the National Institutes of Health Guide for handling laboratory animals and the protocol was approved by the Institutional Animal Care and Use Committee of the Ohio State University. C57BL/6 mice aged 8–10 weeks were used. The 10 wt% hydrogel solutions were sterilized under UV light for 30 min. Before injection, the solutions were precooled to 4°C. Injections were made into the back of each mouse (200 μl/injection and 2 injections/mouse). Two mice were used for each hydrogel type. The mice without injection were served as control. After 4 weeks of injection, tissues were collected and fixed in 4% paraformaldehyde for 48 h. The tissues were then dehydrated, embedded in paraffin, and sectioned into 4 µm thick slices. The sections were then stained with H&E. For immunohistological study, the tissue sections were blocked by 3% BSA, permeated with 0.2% Triton X-100, and incubated with primary antibody of F4/80 overnight under 4°C. After rinsing with PBS, the Alexa fluor 488 secondary antibody was added and incubated for 1 h. Finally, Hoechst was used to counterstain nucleus. Immunofluorescence images were recorded by Olympus Filter FV1000 confocal microscope. The ratio of F4/80 positive cells was quantified from the images.

Cryosections were fixed with −20°C methanol for 5min before immunostaining. Sections were permeabilized with 0.1% Triton X-100 (Sigma Aldrich), blocked with 1% bovine serum albumin, and incubated with primary antibodies overnight at 4°C. The following day, sections were incubated in secondary antibodies for 2 hours at room temperature and mounted in ProLong® Gold Antifade Mountant with DAPI (Life Technologies). The primary and secondary antibodies include: mouse anti-HCN1 (1:100, Abcam), rat anti-HCN4 (1:100, Abcam), rabbit anti-HCN2 (1:100, Alamone), rabbit anti-Cx43 (1:400, Sigma-Aldrich), mouse anti-α-actinin (1:200, Abcam), goat anti-rabbit Alexa Fluor 488 (1:200, Life Technologies), and goat anti-mouse Alexa Fluor 568 (1:200, Life Technologies). Paraffin sections were dewaxed and heated in citrate-based buffer for antigen retrieval before the immunostaining protocol. Sections were imaged using an Olympus FV1000 Filter confocal microscope and florescence density was measured by ImageJ software. A summary of antibodies used in immunohistochemistry protocols is provided in Supplemental Table I, and the specificity of antibody is discussed in Supplemental Material (Supplemental Figure I).

The following protocol was modified from a previous report.^{35 (link)} Cells were seeded on 18 mm glass coverslips in 12-well dishes at 60 000 cells per well. After 24 h, the cells were transfected using Lipofectamine 2000 according to the manufacturer’s instructions. After 24 h, the cells were washed with cold PBS and treated with hypotonic lysis buffer for 8 min on ice. The cells were then washed with cold PBS and immediately fixed using 4% formaldehyde in PBS at room temperature for 10 min. The coverslips were then washed with PBS and mounted in VectaShield Mounting Medium with DAPI stain (Vector Laboratories). The cells were observed using an Olympus FV 1000 Filter Confocal microscope. Images were collected using Olympus Fluoview Ver.2.0b software at room temperature through a UPLFLN 40X Oil-immersion 1.30 numerical aperture objective. Images were converted directly to tiff file format without further processing. Representative images after colorization using Olympus Fluoview Ver.2.0b software are shown (Figures 5A, S5, S6, and S7).