Iba 1

First, 4% paraformaldehyde (PFA) was used to dissect and pre-fix retinas overnight at 4 °C. PBS containing 1% bovine serum albumin (BSA) and 1% Triton X-100 was used to block and permeabilize the samples overnight. We incubated samples with anti-CD31 (BioLegend Cat# 102502, RRID:AB_312909), anti-gal3 (Abcam Cat# 2401-1, RRID:AB_1267156), Iba1 (FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504), Jag1 (Santa Cruz Biotechnology Cat# sc-390177, RRID:AB_2892141) in PBS containing 1% BSA and 1% Triton X-100 overnight at 4 °C, followed by incubation with Alexa Fluor®647 goat anti-rabbit immunoglobulin G (IgG; H + L) secondary antibody (1:100) and Alexa Fluor®594 donkey anti-rat immunoglobulin G (IgG; H + L) secondary antibody (1:100) in PBS. DAPI staining was performed according to the manufacturer’s instructions. Between each step, PBS was used to wash the samples three times for 10 min. Retinas were flat-mounted under a dissecting microscope (Olympus) and a confocal laser scanning microscope (FV1000, Olympus) was used to take photographs. Antibody reagents are listed in Supporting Table S2.

After perfusion, isolated brain was dehydrated overnight in 30% sucrose and embedded in OCT solution (Thermo-Fisher Scientific). To characterize the DREADD receptor expression in the brain and assess if it overlaps with non-neuronal cells, 30-μm sections were blocked for 2 hours in 5% BSA in 0.3% Triton X-100 at RT. Subsequently, sections were incubated overnight at 4°C with primary antibody mix in blocking buffer (Iba-1, 1:1000, Fujifilm, Cat. No. 019-19741). Afterward, cryosections were washed in PBS, incubated with secondary antibody mix in 3% BSA in 0.02% Triton X-100 for 1 hour at RT (chicken anti-rabbit Alexa 647, 1:1000, Invitrogen, Cat. No. A21443) and mounted using Aqua PolyMount (Polysciences). Confocal images were acquired on a Zeiss LSM 510 Meta confocal microscope and AxioCam HRm camera and analyzed using a 10× objective. Sections were imaged and analyzed using ImageJ.

The local immune response to treatment was assessed by characterizing immune cell infiltrates within brain sections collected from high-dose H-FIRE ablation groups at the study endpoint. Tissue samples were sectioned and stained for presence of T cells (CD3, Dako), helper T cells (CD4, Origene), cytotoxic T cells (CD8, Invitrogene), regulatory T cells (FoxP3, Invitrigene), B cells (CD79a, Santa Cruz), microglia (IBA-1, FUJIFILM), M1 macrophages (CD86, Abcam), and M2 macrophages (CD163, Abcam). Methods were followed as presented by Koshkaki et al. (35 (link)). Samples were categorized as peritumoral region (healthy tissue), transition zone (submillimeter zone between the tumor mass and the peritumoral region), tumor mass, and the necrotic core. Tissue samples were imaged using a Nikon Eclipse Ni-U microscope using a Ds-Ri2 camera. The acquired images were analyzed using the NIS element BR software version 5.21.01.
Briefly, auto thresholding was done on all images followed by greyscale conversion. Mean gray values (MGVi) and gray area fractions (AF) were calculated using the software. The values were then used to find the final chromogen intensity (f) as follows:
Mean f and AF values were next calculated using five high power fields from each slide. The immunohistochemistry (IHC) score was calculated using the mean values and the following equation:

Mice were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) and tissue was paraffin embedded. One-micrometre thick slices were stained with haematoxylin and eosin and Luxol fast blue/periodic acid-Schiff. T cells, plasma cells, macrophages and microglia were detected by immunohistochemistry with an avidin–biotin technique using antibodies specific for CD3 (SP7; DCS Innovative Diagnostik-Systeme), IgG (polyclonal; Merck), Mac-3 (M3/84; BD Biosciences) and Iba1 (polyclonal; Fujifilm), respectively. Histological sections were captured using a digital camera (DP71; Olympus Europa) mounted on a light microscope (BX51; Olympus Europa). The percentage of demyelinated white matter was calculated using cellSens Dimension software (Olympus Europa). Inflammatory cells were quantified at × 400 magnification using an ocular counting grid and are shown as cells/mm². At least eight spinal cord cross sections per animal were taken for each analysis.

Pinna samples were collected by the postmortem punch biopsy of the skin (see above) and fixed overnight in 10% neutral buffered formalin, routinely processed, embedded in paraffin, sectioned at 5 μm and stained with Hematoxylin and Eosin (H&E). Immunohistochemistry analyses with antibodies against macrophages (Iba-1; FujiFilm, 019-19,741), eosinophils (MBP; Mayo Clinic, MR-14.7), CD4 (SYSY, HS-360 017), CD8 (SYSY HS-361 003), and Ki67 (Abcam, ab16667) were performed on the Leica Bond Max (Leica Biosystems Inc. Buffalo Grove, IL) following standard protocols in the Translational Pathology Shared Resource at Vanderbilt University Medical Center. Briefly, all assays were antigen retrieved using an Epitope Retrieval 2 solution for 20 min except MBP (Epitope Retrieval 1 solution for 15 min). Leica polymer was used as a secondary antibody for Iba-1, CD8, and Ki67 while a rat secondary antibody (Vector BA-4001) was used for MBP and CD4. The Bond Polymer Refine Detection system was used for visualization. Immunohistochemical stains were evaluated by a veterinary pathologist (K.N.G-C and K.L.B) blinded to the composition of the groups.

Fixed spinal tissue sections were blocked and permeabilized for 1 h with 10% normal donkey serum and 0.3% Triton X-100. The sections were then incubated overnight at room temperature (RT) with mouse monoclonal antibody for ED1 (1:300; Bio-Rad Laboratories, Hercules, CA, USA) and Iba-1 (1:500; Fujifilm Wako Chemical. Richmond, VA, USA). After washing with phosphate-buffered saline (PBS) 2 ml, the slides were incubated for 1 h at RT with fluorescent (Alexa 488 and 594) donkey anti-mouse secondary antibody (1:1000; Life Technologies, Carlsbad, CA, USA). The slides were briefly rinsed with PBS 2 ml and coverslipped with VECTASHIELD containing 40,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). The stained spinal tissue sections were photographed using the BioRevo fluorescence microscope BZ-9000 Generation II (Keyence, Itasca, IL, USA). Fluorescence was measured using BZ-9000 Generation II analyzer (Keyence) and analyzed by NIH ImageJ. Proportional area measurements were acquired by adjusting the thresholds of stained sections in image J and ratio of immunoreactive area to the section area was calculated. Every eighth section of the spinal cord was analyzed for a distance of up to 5 mm in the rostral and caudal directions from lesion center.