Sample size. Sample sizes were dependent on the assay and are presented within the methods section for each assay.

Rules for stopping data collection. Data were collected at a predefined endpoint for each in vitro assay based on manufacturer’s protocol or recommendations from literature. With murine survival studies, mice were euthanized in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

Data inclusion/exclusion criteria. Data generated for all assays were presented.

Outliers. No outliers were removed in this study.

Selection of endpoints. Study endpoints were predefined on the basis of the assay (please see methods sections below) and were not changed on the basis of outcomes.

Replicates. All in vitro assays were performed in multiple replicates as described within the methods section. Murine studies were powered as described below to measure statistically significant differences between groups.

Research objectives. The overall goal of this proof-of-concept study was to identify VLRs that bind normal brain ECM and demonstrate the functional ability of these VLRs to treat neurological pathologies that present with pathologic exposure of brain ECM.

Research subjects or units of investigation. NOD-SCID and C57BL/6 mice, bEnd.3 and 3T3 tissue-cultured cells, U87 and GL261 GBM cell lines, murine tissue sections (multiple organs), human brain sections, and human GBM sections were used in this study.

Experimental design. This study was a controlled laboratory experiment. Initial measurements were made using quantitative ELISA-based assays. Clones were validated using qualitative and quantitative in vitro assays and qualitative and quantitative murine studies.

Randomization. Mice were randomized into groups after intracranial implantation of U87 or GL261 GBM cells.

Blinding. Researchers were not blinded in this study given the limited number of individuals approved to perform murine assays making blinding impractical for this proof-of-concept study.

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