Sl confocal microscope

The analyzed P0 brains (born E19) from Intron-RED + pRS003 (n = 3) and pre-miR-128-2-RED (n = 4) electroporated animals were from the same litter. 60-µm sections were stained for dsRed (Abcam ab62341 1:150) and GFP (Abcam ab13970 1:500). Nuclear staining was obtained with DRAQ5 (Biostatus, Shepshed, United Kingdom). Images were taken using a Leica SL confocal microscope. The overview was taken as a single image with 10× objective. Images for reconstruction of migrating neurons were taken with a 40× objective and a 1 µm step Z-stack. The deep layers were defined using nuclear stain and a pool of migrating neurons within the deep layers was reconstructed using the Fiji plugin Simple Neurite tracer. The number of branches and filopodia (excluding the trailing process) was counted. To distinguish between branch and filopodium a cut-off of 5 µm was used.

Gain‐ and offset‐matched images were collected on a Leica SL confocal microscope in the W.M. Keck Microscopy Facility at the University of Washington. All z‐stacks were collected in 1 μm thick sections. To determine the level of nonspecific staining, sections were incubated without primary antibody and the immunocytochemical reaction was then carried out as described above. Sections stained in the absence of primary antibody showed no detectable labeling. A threshold function was applied to all z‐stack images, which were hand traced using a Bamboo Create digitizing tablet interfaced with Fiji Image J. Motoneuron size and staining density measurements were taken using the Region of Interest (ROI) function in Fiji Image J, which compiled traced outlines of the neuron soma. Each soma was measured for cross‐sectional area, mean stain intensity (8‐bit, max intensity), and roundness, a measure of circularity ranging from 0 to 1 (perfect circle). Roundness measurements were generated using the fit ellipse function on Fiji Image J and selecting “shape descriptors” in the measurement options panel.

Livers were fixed in 4% paraformaldehyde overnight, and tissues were frozen in optimum cutting temperature compound for cryosectioning. IF was performed using standard techniques, with liver sections incubated overnight with the primary antibodies listed in Table S1. Immune complexes were detected with goat Alexa 633 conjugated anti-rat IgG (A-21094, Life Technologies) and goat Alexa 546 conjugated anti-rabbit IgG (A-11010, Life Technologies) antibodies. Sections were mounted with SlowFade Gold (S36936, Life Technologies) and imaged with a Leica SL confocal microscope (Leica Microsystems, Keck Center UW). For ex-vivo imaging, freshly harvested livers were analyzed as previously described [27] . Images were captured using a Zeiss 510 Meta confocal microscope. In Pdgfrα^WT/nGFP mice, GFP fluorescence was used to report PDGFRα positive cells [20] (link).

The liver from C57BL/6 mice were dissected and fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) at 4°C, overnight. The tissue was then cryoprotected with series of sucrose gradient (10%, 20% and 30% w/w) in phosphate buffer at 4°C and cut into 20-μm sections. The sections were washed 3 times in phosphate-buffered saline (PBS), blocked in PBS containing 5% goat serum, 1 mg/mL bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 hour at room temperature and incubated with monoclonal rat anti-CXCL1 (1:100; clone: 124014, R&D), polyclonal rabbit anti-CRBP1 (1:50; FL-135, Santa Cruz) or Alexa Fluor 647-conjugated rat anti-F4/80 antibody (Serotec) in PBS containing 1mg/mL BSA and 0.1% Triton X-100, at 4°C, overnight. The sections were washed and stained with Alexa Fluor 546-conjugated goat anti-rat IgG (for CXCL1) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (for CRBP1) (1:500; Invitrogen). In genetically modified mice, GFP expression under Tie-2 promoter restricted GFP signal to LSECs. Sections were stained with TO-PRO-3 (1:1000; Invitrogen) for nuclear visualization in HSCs. All sections were mounted with SlowFade Gold antifade reagent (Invitrogen) and observed with a Leica SL confocal microscope under a 100X objective lens (Leica Microsystems) located in the Keck Imaging Facility at the University of Washington.