Vectashield medium with dapi

Marrow cells were cultured in IMDM with 20% FBS, murine SCF (100ng/ml), and IL-6 (200ng/ml) for two days. Cells were then exposed to 0.2 μg/ml colcemid (Life Tech) for 4 hours and pelleted (800 rpm) for 5 minutes. Cells were resuspended dropwise in pre-warmed (37°C) 75mM KCl while vortexing gently and incubated at 37° C for 15 minutes. After pelleting, cells were resuspended in a 3:1 methanol: glacial acetic acid. Cells were pelleted and resuspended in fixative solution two additional times before being dropped onto slides and dried overnight. Spreads were then mounted in Vectashield medium with DAPI (Vector Laboratories). For the chromosome breakage test, cells were cultured for 48 hours in 50nM MMC before colcemid addition. For spectral karyotyping (SKY), samples were imaged and analyzed by the MD Anderson Cancer Center Molecular Cytogenetics Facility (Houston, TX).

We cultured primary microglia on poly-D-lysine (PDL)-coated cover slips until DIV 14. Microglia were washed with ice-cold PBS and fixed in ice-cold methanol for two minutes. This was followed by three washes for five minutes each with ice-cold PBS. We then permeabilized the microglia with 0.03% Triton X-100 dissolved in PBS for four minutes. Next, cells were blocked with 10% normal goat serum for 1 h at room temperature, followed by incubation with primary antibody IBA1 (1:500, #019-19741, Wako), CX3CR1 (1:250, #14-6093-81, Thermo-Fisher) TREM2 (1:250, #76765S, Cell Signaling) diluted in 10% normal goat serum in PBS. Cells were then incubated in primary antibody overnight at 4 °C. The following day cells were washed with PBS three times for five minutes and secondary was added, goat anti-mouse Alexa Fluor 568 secondary antibody (1:2000, #A11031, Thermo-Fisher) diluted in 10% normal goat serum in PBS. The cells were incubated in secondary antibody for 2 h at room temperature, washed with PBS three times for five minutes, and coverslipped with Vectashield medium with DAPI (Vector Laboratories).

Mice were euthanized and perfused with ~50 ml of PBS. Brain tissue was extracted and fixed in 4% PFA (pH = 7) for 24 h at 4 °C. PFA was washed three times with PBS, and brain tissue was cryoprotected in 30% sucrose dissolved in PBS for 94 h at 4 °C. 10 μm frozen coronal serial sections were cut on a cryostat and mounted on polarized glass slides (Fisherbrand Superfrost Plus microscope slides, #12–550–15, Fisher Scientific, Waltham, MA). We removed OCT by washing slides in PBS for 10 min. Tissue was permeabilized by incubating the slides with 3% Triton-X dissolved in PBS for 10 min. Slides were washed three times for 5 min each in PBS and probed with experiment specific primary antibodies: Synaptophysin (1:500, #ab32127 Abcam), Iba1 (1:500, #019–19741, Wako), or Iba1 (1:250, #MABN92, EMD Millipore). The slides were incubated overnight at 4 °C in primary antibody, followed by washing PBS and incubation with specific secondary antibodies for 2 h: goat anti-mouse Alexa Fluor 568 (1:2000, #A11031, Thermofisher) and goat anti-rabbit Alexa Fluor 488 (1:2000, #A11008, Thermofisher). Post incubation, slides were washed and mounted in Vectashield medium with DAPI (Vector Laboratories), coverslipped, and sealed with nail polish.

Experimental mice were perfused with 4% paraformaldehyde (PFA) and dissected brain specimens were post-fixed for an additional 4 h at room temperature. Brains were washed three times in PBS, then submerged in a 30% sucrose solution and allowed to sink at 4 °C before embedding in OCT compound (Tissue-Tek) and cutting. 20-µm coronal sections were placed onto Superfrost Plus slides (Fisher Scientific), permeabilized with 0.01% Triton XT-100 in PBS and blocked with 5% normal goat serum (NGS) in PBS at room temperature for 1 h. Primary antibodies diluted either 1:100 (IFNAR1) or 1:250 (GFAP, NeuN, OLIG2, CD11b, IBA1) in 1% bovine serum album (BSA) solution were applied onto the sections overnight at 4 °C. After extensive washing, the sections were incubated with secondary antibodies diluted 1:500 in 1% BSA for 1 h at room temperature in the dark. Finally, coverslips were mounted using Vectashield medium with DAPI (Vector Laboratories) and sealed with nail polish. Prepared slides were imaged at the Brody School of Medicine Cell Analysis Core using an LSM 700 confocal microscope (Zeiss) and analyzed using ImageJ software. For quantitative fluorescence measurements, the exact same lens, pinhole, laser, and detector settings were used to image mock injected control specimens and experimental EAE tissue specimens.

Apoptotic cells were detected in paraffin sections of fetal testes using the Fluorescein in situ Cell Death Detection Kit (Roche Applied Science) and mounted in Vectashield Medium with DAPI (Vector Laboratories).