Axiovert 40 cfl fluorescent microscope

F-actin ring formation was visualized by phalloidin staining as previously described with minor modifications 38 (link). In brief, BMMs were washed with PBS prior to fixation with 4% paraformaldehyde for 10 min at RT. The fixed cells were then permeabilized with 0.1% Triton X-100 for 5 min, followed by incubation with 3% BSA in PBS for 20 min. Subsequently, cells were stained with FITC-conjugated phalloidin (1:100) at RT in darkness for 20 min. After washing, cells were stained with DAPI (1:2000) for 5 min, rinsed with PBS, and then mounted with anti-fade mounting medium (Beyotime Biotechnology, Shanghai, China). Fluorescent staining was observed and photographed using the Axiovert 40 CFL fluorescent microscope installed with Zeiss ZEN software (Carl Zeiss Microscopy, Thornwood, NY). The number and relative size of F-actin rings in each group were measured using Image J software (National Institutes of Health, MD, USA). A minimum of 3 wells per group were analyzed.

Primary culture of mouse brain endothelial cells were isolated from adult male CD-1 mice (8 weeks old) as previously described [21 (link)]. Brain microvessel endothelial cells were treated with or without epinephrine (10 μM) in serum-free DMEM/F-12 (Sigma) supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin, 2 mM GlutaMAX^™-I and 1 ng/mL basic fibroblast growth factor (bFGF; Sigma) for 30 min. After fixation with 3.7% formaldehyde (Sigma) for 10 min at room temperature, they were incubated with anti-M6P receptor antibody (Abcam) in 1% BSA/PBS for overnight at 4°C. Then, they were washed once with PBS, three times with balanced salt solution (130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 4 mM MgCl₂, 20 mM HEPES, 5.5 mM glucose, pH 7.4) and once with PBS, and incubated with 20 μg/mL Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen) in 1% BSA/PBS for 1 hr at room temperature. After washing, cells were covered with Vectashield Hard Set mounting medium (Vector Laboratories, Burlingame, CA) and coverslips were applied. Fluorescence was detected with Zeiss Axiovert 40 CFL fluorescent microscope.