The VLR YSD library underwent two rounds of biopanning against bEnd.3 ECM to enrich for brain ECM-binding clones as previously described (20, 42). For the ELISA-based screen, selected biopanning clones were streaked out, and individual clones were picked into 96-well polypropylene plates. Clones were expanded at 30°C overnight in SD-CAA medium and split into two plates (one plate for screening and one plate for frozen stock). The screening plate was induced at 20°C using SG-CAA medium for 24 hours. VLR displaying yeast were washed with 50 mM HEPES (pH 7.2), and VLR was reduced off of the yeast surface using 20 μl of 50 mM MESNA solution in 50 mM HEPES (pH 7.2) for 45 min. Supernatants containing VLR were then diluted 1:10 with 50 mM HEPES (pH 7.2). Plates containing decellularized bEnd.3 or 3T3 ECM were blocked with 1% BSA and 1.5% goat serum in PBS and incubated with the HEPES-diluted VLRs for 1 hour at 37°C. Wells were washed five times with PBS + 0.05% Tween 20. Next, an anti-myc antibody (9E10, BioLegend) modified with horseradish peroxidase (HRP) (1:1000) was added to each well and incubated for 45 min at room temperature. Wells were then washed seven times with PBS + 0.05% Tween 20, 1 min per wash. VLR binding was detected by incubation with one-step 3,3′,5,5′-tetramethylbenzidine (TMB) substrate for 15 to 30 min to develop signal. The reaction was stopped via acidification with 1 M HCl and quantified using absorbance signal at 450 nm. A blank well was included on every plate to establish background signal.

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