Alex fluor 488 secondary

Brain endothelial cell (BEC) expression of claudin-5 and ZO-1 was examined as described previously [36 (link)]. Briefly, BECs derived from the β4-EC-KO or littermate control mice were first passaged onto laminin-coated six-well plates. Upon reaching confluence, the cells were treated with either 10 ng/ml TNF-α or IFN-γ (both from R&D) for 48 h and then removed and cellular expression of claudin-5 and ZO-1 analyzed by flow cytometry using rabbit polyclonal anti-claudin-5 or anti-ZO-1 antibodies followed by anti-rabbit Alex Fluor 488 secondary (both from Invitrogen). As the claudin-5 and ZO-1 antibodies are directed against intracellular epitopes, the cells were first fixed and then permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen) and all subsequent incubations were performed in the cytoperm buffer. The fluorescent intensity of labeled cells was analyzed with a Becton Dickinson FACScan machine, with 10,000 events captured for each condition. Each experiment was repeated a minimum of four times and the data expressed as mean ± SEM fluorescent intensity. Statistical significance was assessed by using the Student’s paired t test, in which p < 0.05 was defined as statistically significant.

Whole blood from healthy females over the age of 18 years was purchased from Innovative Research (Novi, MI). Monocytes were enriched by negative selection using the Rosette Sep^® monocyte enrichment cocktail according to manufacturer's instructions (STEMCELL Technologies; Vancouver, Canada). To differentiate isolated monocytes into the M2 phenotype, 9 mm square coverslips were placed in individual wells of a 24 well plate and monocytes were seeded at a density of 200,000 cells/well for 6 days in AIM V media (Invitrogen) supplemented with 1% penicillin-streptomycin in the presence of 20 ng/mL M-CSF (Peprotech; Rocky Hill, NJ). Macrophages were then activated for 48 hours in 2 ng/mL each of IL-4 and IL-13 (Peprotech). Phenotypical characterization of MDM was performed by immunofluorescence after 8 days using anti-CD68 (clone Y1/82A), anti-CD206 (clone 19.2), and their respective isotypes (BD Biosciences; Franklin Lakes, NJ) with a goat anti-mouse Alex Fluor 488 secondary (Invitrogen). Fixed cells were imaged at room temperature in phosphate buffered solution (PBS, Invitrogen) on a Zeiss Axio Observer.Z1 inverted microscope with an AxioCam 506 mono camera, a Plan-NEOFLUOR 20x 0.4-NA air objective, and Zen2 software (Zeiss; Oberkochen, Germany).