Aprochromat objective

Ten random fields per treatment condition were captured using a 63× oil-immersion Aprochromat objective (Carl Zeiss) and the total pixel values for each antibody immunofluorescence staining (CD44, CSPG, and decorin) were measured using Axiovision software. The experimental conditions were done in triplicate and repeated three times independently and the quantifications were done by an operator blinded to the treatments.

Imaging was performed with a Carl Zeiss Axiovert M1 microscope with 100x Plan Aprochromat objective. Images were taken with a Hamamatsu Orca-ER charge-coupled device and processed with MetaMorph, version 7.1 software (Molecular Devices). L4 hermaphrodites were used for all imaging. Animals were paralyzed with 30 mg/mL 2, 3-butanedione monoxamine (BDM, Sigma-Aldrich) for 5 minutes before imaging. For quantitative VNC imaging, maximum intensity Z-series stacks, 1 μm total thickness, were taken from the anterior VNC. FluoSphere fluorescent beads were used to normalize image fluorescence intensity. Line scans were made using MetaMorph (v6.0) and analyzed using IgorPro (v5) and custom-written software [52 (link)]. Constant exposure settings were used across genotypes and across all imaging days. Cell counts of GLR-1-expressing neurons marked with Pglr-1::NLS-GFP-LacZ (pzIs29), NMR-1-expressing neurons marked with Pnmr-1::GFP (akIs3), ASH neurons stained with DiI and ASH neurons marked with ChR2::YFP (ljIs114) were done manually by focusing through the worm head using a 63X objective.