Fluoromount g antifade reagent

For immunofluorescence, tissues were immediately isolated, fixed for 2 h with 4% paraformaldehyde in PBS at 4 °C and soaked in 30% sucrose in PBS overnight at 4 °C. Tissues were then embedded in Tissue-Tek OCT blocks (Sakura) and frozen in liquid nitrogen. The frozen samples were sectioned at a thickness of 10 μm by cryostat (Leica, CM3050S). The following antibodies were used: anti-mouse CD3ε (BioLegend, clone 145-2C11), anti-mouse B220 (eBioscience, clone RA3-6B2) and anti-mouse CD11c (SouthernBiotech, polyclonal). Tissue sections were counterstained with DAPI (Sigma-Aldrich) and mounted with Fluoromount-G antifade reagent (SouthernBiotech).
For human tissue staining, tissues were freshly isolated, embedded in Tissue-Tek OCT blocks (Sakura) or SCEM (SECTION-LAB) and frozen in liquid nitrogen. The frozen samples were sectioned at a thickness of 10 μm by cryostat (Leica, CM3050S). The following antibodies were used for immunohistochemistry staining: anti-human CD68 (eBioscience, clone 815CU17), anti-human CD19 (Abcam, clone EPR5906) and anti-human IgA (SouthernBiotech, polyclonal). Fluorescence images were obtained using a BZ-X700 fluorescence microscope (Keyence).

The spinal cord sections were processed according to the manufacturer’s instructions in the RNAscope Fluorescent Multiplex Assay v2 manual for fixed frozen tissue (Advanced Cell Diagnostics), and coverslipped with Fluoromount-G antifade reagent (Southern Biotech) with DAPI (Molecular Probes)^{47 (link),66 (link)}. The following probes, purchased from Advanced Cell Diagnostics were used: Grpr (nucleotide target region 463-1596; GenBank: NM_008177.2), Tac2 (nucleotide target region 15-684; GenBank: NM_009312.2), Vglut2 (nucleotide target region 1986-2998; GenBank: NM_080853.3), Vgat (nucleotide target region 894-2037; GenBank: NM_009508.2), and Npy1r (nucleotide target region: 227-1169; GenBank: NM_010934.4). Sections were subsequently imaged under a Nikon C2+ confocal microscope (Nikon Instruments, Inc.) in three channels with a 20× objective lens. Positive signals were identified as three punctate dots present in the nucleus and/or cytoplasm if the signal was shown up in small dots rather than filling up entire neurons. For co-localization studies, dots associated with single DAPI stained nuclei were assessed as being co-localized. Cell counting was done by a person who was blinded to the experimental design.

Fluorescent staining was performed using the following antibodies: rabbit anti-Twi (Figeac et al., 2010 (link)) (1:300), rabbit anti-dMyc (1:300) (Santa-Cruz Biotechnology), goat anti-GFP (1:1000) (Biogenesis), rabbit anti-PH3 (1:1000) (Millipore), mouse anti-NICD (1:150), rat anti-Deltex (1:50) (kindly provided by S. Artavanis-Tsakonas, Harvard Medical School, USA), mouse anti-Lamin (1:1000) (DHSB LC28.26), rat anti-Tropomyosin (1–200; Babraham Bioscience Technologies, UK; BT-GB-141), mouse anti-αPS1 (1:50; DHSB; DK.1A4), mouse anti-βPS (DSHB CF.6G11), Phalloidin-TRITC (1:1000) (Sigma). Cy3, Cy5 and Alexa 488-conjugated secondary antibodies (Jackson ImmunoResearch) were used (1:300). Embryos were mounted in Fluoromount-G anti-fade reagent (Southern Biotech). Labeled embryos were analyzed using Leica SP5 and SP8 confocal microscopes. 3D reconstructions of the images were generated using Imaris software (Bitplane).