Sp5 x mp inverted confocal microscope

Cells were incubated with 100 μg/mL human serum albumin (HSA) conjugated to FITC (HAS-FITC) (Abcam) in basal DMEM/F12 medium for 1 h at 37°C. For negative control, cells were incubated with HSA-FITC at 4°C. The cells were fixed with 4% paraformaldehyde and then counterstained with DAPI. Samples were visualized with a Leica SP5 X MP inverted confocal microscope and the images were processed in ImageJ.

The astrocytes were incubated in 4% paraformaldehyde for 15 minutes at room temperature and washed several times using HBSS supplemented with 10 mM of CaCl₂. For antibody staining, the cells were permeabilized with 0.1% Triton X-100 in HBSS for 8 minutes, washed 6 times with HBSS with added calcium, and then incubated with blocking buffer (5% goat serum, 1% bovine serum albumin). Then, the cells and gels were incubated with an antibody against GFAP (Abcam, ab33922, concentration 1:300), MAP2 (Invitrogen, PA1-10005, concentration 1:5000), NeuN (Abcam, ab104224, concentration 1:1000), β3-tubulin (Abcam, ab18207, concentration 1:2000) overnight at 4°C. The next day, the samples were incubated with a fluorescently labelled secondary antibody (Alexa488, Abcam 1:500; goat anti-chicken Alexa 555 ab150170 1:500; goat anti-mouse Alexa 488 Invitrogen A-11029 1:500; goat anti-rabbit Alexa 555 ab150078 1:500) for 1 hour at room temperature. Staining for cell nuclei was performed with Hoechst 33342 (concentration 1:1000) for 10 minutes, at which point the samples were transferred to slides. Mounting media (Prolong Gold Glass Antifade, Invitrogen) was added, and a coverslip placed on top of the gels. Images were taken with a Leica SP5 X MP Inverted Confocal microscope at 4x, 10x, 20x, 40x oil, and 63x oil magnification. More than 10 random fields were taken per sample.