Fluorescence microscope

Male and female mice (n=2 each sex, 8–12 weeks old) were used to verify mRNA expression in the VTA using RNAscope. Brains were flash-frozen in 2-methylbutane and representative coronal sections that spanned the VTA were sliced at 20 µm and slide mounted for hybridization. Sections were prepared for hybridization per the manufacturer’s (Advanced Cell Diagnostics, Inc) instructions using probes for Th (Mm-Th), Ntn1 (Mm-Ntn1-C2), and Slc32a1 (Vgat; Mm-Slc32a1-C3). Slides were coverslipped with Fluoromount with DAPI (Southern Biotech) and imaged using a confocal fluorescent microscope (the University of Washington Keck Center Leica SP8X confocal) and Keyence Fluorescence Microscope (Keyence). Quantification of co-labeled cells was performed using CellProfiler, with thresholding and cell identification/overlap for each channel verified for each image manually prior to quantification.

Lungs and LNs were excised and immersed in 4% paraformaldehyde with 10% sucrose and 7% picric acid in PBS for 2 hr and then embedded in Tissue-Tec OCT (Thermo Fisher Scientific). 10 µm sections were prepared and slides were stained for B220, SiglecH, and IFN-α. Control slides were treated with Ab isotype controls and secondary antibodies. Stained slides were mounted with Prolong Gold Antifade containing DAPI (# P36931, Invitrogen), whole lymph node images were stitched using 10× objective lens and imaged with a Keyence fluorescence microscope (BZ-X800 series).

Mice were anesthetized with pentobarbital and transcardially perfused with PBS followed by 4% PFA. Brains were post-fixed for 24 hr in PFA at 4°C, followed by 48 hr in 30% sucrose. The VTA was coronally sectioned at 30 µm. Sections were kept in PBS with 0.3% Sodium Azide. Free-floating sections were treated with 0.3% TBS-Triton-X 100 3x10 min, blocked in 3% Normal Donkey Serum for 1 hr and treated overnight in primary antibody. Following 1–3 hr in secondary antibody (JacksonImmuno), sections were slide mounted and coverslipped with Fluoromount with DAPI. Images were collected on a Keyence Fluorescence Microscope (Keyence). For CRISPR validation, male and female DAT-Cre mice (8–12 weeks old) received AAV1-FLEX-SaCas9-U6-sgNtn1/AAV1-FLEX-YFP (Ntn1-cKO) or AAV1-FLEX-SaCas9-U6-sgROSA26/AAV1-FLEX-YFP (controls) injections as described above, and quantification of co-labeled cells for immunohistochemistry were performed using ImageJ 1.53 Cell Counter/Multi-point tool. Total Th-positive cells were recorded for all images and averaged across all slices for each mouse to give a total number of Th-positive cells. Primary antibodies used: mouse anti-TH (1:1500, Millipore), chicken anti-Netrin-1 (1:1000, Abcam), and rabbit anti-HA (1:1500, Sigma).

Mutant GFP-exon 1-based aggregation was monitored with the Keyence fluorescence microscope. A total of 24 h after transfection cells very fixed using 4% PFA. When using GFP-exon 1 variants either with or without enzymatically active TG6, at least 200 (double) transfected cells were counted per coverslip and the proportion of cells with at least one aggregate/inclusion was scored as a percentage of the total number of transfected cells. The experiments were performed without knowing the identity of the slides at three times in triplicates.
Presented data are mean ± SEM of three independent experiments counting at least 200 cells per coverslip using the Prism6 software. For statistical analysis two-way ANOVA and Bonferroni post-test were used.

Fixed or live cells were imaged using a confocal microscope LSM710 (Carl Zeiss, Inc.) or Keyence Fluorescence Microscope (BZ-X700). Immunofluorescence was performed as previously described [9 (link)]. Briefly, WI-38 fibroblasts were fixed with 100% methanol (Fisher Scientific) or 3.7% formaldehyde (methanol-free, Polysciences) for 10 min at -20 °C or RT, respectively, and permeabilized in 0.2% Triton X-100 for 5 min. After washes with PBS and incubation with 10% normal goat serum (Thermo Fisher Scientific) for 16 h at 4 °C, anti-GRSF1 (Sigma) or anti-53BP1 (BD Biosciences) antibodies were added to the cells and incubated at 37°C for 1 h. Washed cells were then incubated at 37 °C for an additional 30 min with Alexa Fluor 568-conjugated secondary antibodies (Thermo Fisher Scientific). Nuclei were stained in ProLong® Gold Antifade reagent with DAPI (for fixed cells, Thermo Fisher Scientific) or Hoechst 33342 dye (for live cells, Invitrogen). To measure the formation of mitochondrial superoxide, WI-38 fibroblasts were stained with a fluoroprobe (MitoSOX Red, Thermo Scientific) and assessed by the Keyence Fluorescence Microscope (Keyence).