Goat anti mouse af568

Polyclonal rabbit antibodies were raised against full‐length human Munc18‐2 (5182 and 5184) and STX11ΔCys‐GST (5412 and 5413) as previously described 25. Other antibodies were sourced: mouse anti‐human LAMP1 [H4A3] (Iowa University Hybridoma cell bank), mouse anti‐human CD8 [UCHT4] (Sigma‐Aldrich, C7423), mouse anti‐human perforin [δG9] (BD Pharmingen, 556434), mouse anti‐beta actin [AC‐15] (Sigma‐Aldrich, A5441), mouse anti‐human LAMP1‐PE [H4A3] (eBioscience, 12–1079), mouse anti‐human CD8‐APC [MEM‐31] (Abcam, ab26004), mouse anti‐human CD4‐PE [MEM‐241] (Abcam, ab18282). Secondary antibodies goat anti‐rabbit‐AF488 (A11034, highly cross‐adsorbed) and goat anti‐mouse‐AF568 (A11031, highly cross‐adsorbed) and Hoechst nuclear stain 33342 were from Invitrogen. HRP‐conjugated secondary antibodies used for immunoblotting were goat anti‐rabbit IgG (Jackson, 111‐035‐144) and goat anti‐mouse IgG (Jackson, 113‐035‐146). Ionomycin and PMA were obtained from Sigma‐Aldrich.

For immunoblotting, mouse anti-myc antibody (Calbiochem) was used at 1:1,000, mouse anti-α-tubulin (Sigma-Aldrich) was used at 1:1,000, rat anti-HA 3F10 (Roche) was used at 1:1,000, goat anti-GFP (Sicgen) was used at 1:1,000, goat anti-MOMP (Abcam) was used at 1:1,000, and mouse anti-CT288 (a gift from Guangming Zhong; Li et al., 2008 (link)) was used at 1:1,000. Horseradish peroxidase-conjugated secondary antibody anti-mouse (GE Healthcare) was used at 1:10,000, anti-rat (Sigma-Aldrich) was used at 1:10,000, and anti-goat (Jackson ImmunoResearch Laboratories) was used at 1:10,000. For immunofluorescence, mouse anti-γ-tubulin antibody (Sigma-Aldrich) was used at 1:200, mouse anti-CT442 antibody (a gift from Guangming Zhong; Li et al., 2008 (link)) was used at 1:200, rat anti-HA 3F10 antibody (Roche) was used at 1:200, goat anti-GFP antibody (Sicgen) was used at 1:200, and goat anti-MOMP antibody (Abcam) was used at 1:200. Secondary antibody donkey anti-goat conjugated to cyanine 5 (Jackson ImmunoResearch Laboratories) was used at 1:200, anti-rat conjugated to rhodamine RedX (Jackson ImmunoResearch Laboratories) was used at 1:200, and goat anti-mouse AF568 (Invitrogen) was used at 1:200. 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) was used to stain DNA.

Brains and spinal cords of mice with EAE were dissected. Mice were either perfused and tissues fixed in 4% paraformaldehyde (PFA) and paraffin embedded, or tissues were embedded in OTC compound (Sakura Finetek Denmark ApS, Værløse, Denmark) and snap-frozen in isopentane on dry ice. Tissues were sectioned in 6–10 μm slices, were fixed in 4% Paraformaldehyd (PFA) for 10 min, either stained with haematoxylin and eosin (H&E, Histolab) staining or different antibodies were used for staining and visualized. Primary antibodies used for immunofluorescence (IF) were as follows: rabbit anti-TCR alpha (Abcam, ab18861, 1:30), rat anti-PDL1 (Abcam, ab80276, 1:200), mouse anti-FoxA1 (Millipore, 05-1466, 1:2,000) and rabbit anti-NF200 (Sigma, N4142, 1:50). Secondary antibodies to IgG used were as follows: goat anti mouse-Af568 (Invitrogen, A11031), goat anti-rat IgG- Af488 (Invitrogen, A11006), goat anti-rabbit IgG- Af633 (Invitrogen, A21071), goat anti-rabbit IgG-Pacific Blue (Invitrogen, P10994), all in concentration of 1:500, Drag5-633 (BioStatus, DR05500, 5 μm) or DAPI-Pacific blue (Invitrogen, D3571, 1:30,000) were used for nuclei visualization. IF images were taken with a Zeiss LSM510 confocal scanning microscope or with IN Cell Analyzer 2200 automated microscope. H&E images were taken with a NanoZoomer 2.0-HT digital slide scanner or Olympus BX51 microscope.