Odyssey laser scanner

Whole cell bacterial lysates were prepared for Western blot analysis as follows. Bacteria were harvested from overnight growth on CFA agar with or without 200 µM DFOM in PBS, and the OD₆₀₀ was used to normalize to the samples for bacterial numbers (55 (link)). The normalized suspensions were then mixed 1:1 with 2× Laemmli Sample Buffer (Bio-Rad). Proteins were separated on a 12% Mini-Protean TGX Precast Gel (Bio-Rad) and transferred to PVDF membrane. The membrane was blocked in a 10% (wt/vol) nonfat milk buffer in PBS and incubated with absorbed polyclonal Rabbit α-CS14 (Rockland Immunochemicals). The secondary antibody was Goat α-Rabbit 680 nm (Invitrogen), and proteins were visualized using the LI-COR Odyssey Laser Scanner. Additional Western blots of CFA agar with or without 200 µM DFOM grown strains were performed and stained with Rabbit anti-DnaK (Invitrogen) and Goat α-Rabbit 680 nm (Invitrogen) antibodies to confirm equal loading.

Blood brain barrier (BBB) permeability was measured using a far-red dye (Alexa Fluor 680, ThermoFisher Scientific, MA, USA) bioconjugated to a 10 kDa dextran molecule, as previously reported by our group and used in several additional studies (39 (link)–42 (link)). CCI and sham injuries were induced as previously described. In this set of injuries, rats were sacrificed 72 hours post-injury. This time point was chosen to keep this study consistent with our prior studies of BBB permeability after injury (41 (link), 43 (link)–45 (link)). At 72 hours after injury and 30 minutes prior to euthanasia, 0.5 mL of 1 mg/mL Alexa Fluor 680 dye was intravenously injected in rats via tail vein. Following exsanguination and 4% paraformaldehyde cardiac perfusion, the brains were harvested, sliced in 2 mm coronal sections and scanned for Alexa Fluor 680 signal using an Odyssey laser scanner (LI-COR Biosciences, NE, USA) in the 700 nm emission channel; auto-fluorescence was visualized in the nonspecific 800 nm emission channel. Quantitative measurements of dye extravasation were determined for each brain by using a uniform region of interest and measuring mean intensity and the mean integrated density of the slices using a threshold to exclude background and low intensity autofluorescence (ImageJ).

Human prefrontal cortex fractions or rat whole brain homogenates were separated on 10% Bis-Tris NuPAGE Novex pre-cast gels (Fisher Scientific, Loughborough, UK) (typically 20 µg/gel lane) run with 3-morpholinopropane-1-sulfonic acid (MOPS) running buffer according to published procedures [10 (link)]. Resolved proteins were either stained with colloidal Coomassie blue and then densitometry performed using an Odyssey laser scanner (LI-COR Biosciences, Lincoln, Nebraska, USA), or proteins electroblotted at 80 V for 2 h to a polyvinylidene difluoride (PVDF) (Millipore, Burlington, MA, USA) membrane for Western blotting. Protein homogenates were typically resolved 3–4 times by gel electrophoresis for each brain sample, with representative images used for Figures. Similarly, protein extracts were resolved 3–4 times for Western blotting analysis, with representative images used for Figure production, and protein level quantitation.