Texas red dextran

To prepare for the Texas Red dextran-labeled cells, 5 x 10⁴ RAW264.7 macrophages were incubated with [10 mg/mL] Texas Red dextran (Invitrogen) in 600 μL DMEM for 18 h at 37°C in 5% CO₂. The Cry3Aa-GFP-labeled cells were prepared by seeding 5 x 10⁴ RAW264.7 macrophages on an 8-well chamber slide and incubated with Cry3Aa-GFP crystals for 2.5 h at 37°C in 5% CO₂. At the end of incubation, the cells were washed with PBS three times to remove excess labeling reagent. The Texas Red dextran RAW cells were then added to the Cry3Aa-GFP RAW cells and incubated for 2 h. At the end of the 2-h incubation period, cells were washed with PBS three times and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The fixed cells were washed three times with PBS before counterstained with DAPI. Cells were washed extensively with PBS and coverslipped using Gel/Mount. For each well, multiple fluorescence images were sequentially taken using an Axioscope 40X microscope in a grid-like fashion to cover the entire cellular population. These images were then manually inspected to tabulate the number of cells containing either Texas Red dextran only, Cry3Aa-GFP crystals only, or both particles.

Beginning at least two weeks after surgery mice were gradually habituated to the imaging rig. Habituation reduces both excessive movement during image collection and chronic restraint stress, where the latter can influence microglial morphology [14 (link)]. In vivo images were collected 12 weeks after surgery, well after astrocytic and microglial responses to the surgery had subsided (30 days after V1 cranial window surgery for astrocytes, and after 7–10 days for microglia [15 (link)]). During imaging sessions, mice were head-fixed underneath a two-photon microscope (Scientifica, Uckfield, UK) atop of a free-moving polystyrene cylinder (Biosciences Workshop, UCL) fitted with a rotary encoder (Kubler Group, Villingen-Schwenningen, Germany) that allowed recording of voluntary running. Imaging sessions were performed in the dark. Immediately prior to imaging sessions, the vascular lumen was labelled either by intravenous injection with 2.5% (w/v) Texas Red Dextran (70 kDa, Invitrogen, Carlsbad, CA, USA) or subcutaneous injection of 2.5% (w/v) Texas Red Dextran (3 kDa, neutral, Invitrogen).

The lyophilized form of TeNT-LC and BoNT/B/D-LC was reconstituted with a solution consisting of 100 mM KCl and 300 µM 3 kDa neutral dextran-Texas red (Life Technologies Corp., Carlsbad, CA) such that the concentration of TeNT-LC and BoNT/B-LC were 0.5 µg/μL and BoNT/D-LC was 0.3 µg/μL. The fluorescent dextran-Texas red dye was used to allow visualization of neurotoxin injection. Control neurotoxin solutions were denatured in a boiling water bath for 30 min.

Osmotic water permeability across endothelial cell layer was determined using Texas Red™-dextran (10 kDa; Molecular Probes, Eugene, OR) dilution method [62 (link)]. bEnd.3 cells were seeded at 1 × 10⁵ cells per insert in Transwell inserts with 0.4 μm pore size (Falcon, catalog No. 353095) and incubated for 2–4 days until cell layer formation. After Au-NP treatment (500 ng/mL; 24 h), the basal and apical surface of cells was bathed in isosmolar PBS and hyperosmolar PBS (PBS with 300 mM D-mannitol) containing 0.25 mg/mL Texas Red-dextran, respectively. Cultures were placed in incubator (37 °C, 5% CO₂), and 5 μl samples of dye-containing apical fluid were collected at 10, 20 and 60 min. The samples were diluted in PBS, and fluorescence was measured by Fluoroskan Ascent™ microplate fluorometer (Thermo Scientific™, Vantaa, Finland).