Eclipse ti2 spinning disk confocal microscope

For the immunohistochemistry analysis, we used the following primary antibodies: rabbit anti-Tamalin (1:200, Novusbio), mouse anti-HuC/D (1:100, Molecular probes), mouse anti-olig2 (1:200, IBL America), and mouse anti-proliferating cell nuclear antigen (PCNA) (1:200, DAKO). To detect fluorescent antibody labeling, we used Alexa 488, 568, 647-conjugated secondary antibodies (1:1000, Molecular Probes). We captured fluorescent pictures of transverse sections using an A1 laser-scanning confocal microscope (Nikon, 1-um z-stack), and wholemount lateral images were obtained using an Eclipse Ti2 Spinning disk confocal microscope (Nikon, 2.5-um z-stack). For in situ RNA hybridization, tamalin and the arf6 open reading frame were cloned into the pGEM T-easy vector (Promega). Not1 and SacII restriction enzyme were used for linearization and transcribed using a DIG labeling combination. Whole-mount or fluorescent in situ RNA hybridization were performed as previously described [36 (link),37 (link)]. The primers were designed using the following sequences:
Tamalin forward: 5′-AGGAGTCCTTTGGCTTCG-3′, Tamalin reverse: 5′-GCTTTCCTCCTCCTCCAGAG-3′, Arf6a forward: 5′-ATTTATGCCCAGCCAAC-3′, and Arf6a reverse: 5′-TTCATTGGCGTTAGGATTTG-3′.

For immunofluorescence, BMEC were subcultured on glass coverslips. Cells were fixed in 4% paraformaldehyde (PFA; Millipore Sigma, P6148) for 20 min, after which they were blocked and permeabilized in 5% normal goat serum (Millipore Sigma, S26) with 0.2% TritonX-100 (Alfa Aesar, A16046) in phosphate buffered saline (PBS; Thermo Fisher, 70011069) for 1 h. Cells were incubated in primary antibodies for GLUT-1 (Thermo Fisher, 21829–1-AP, 1:100), zona occludins-1 (ZO-1; Cell Signaling Technology, 13663S, 1:100), and occludin (Cell Signaling Technology, 91131S, 1:100) overnight at 4°C. Cells were then incubated in a goat-anti-rabbit secondary antibody (Thermo Fisher, A-11012, 1:1000) with Hoechst (Thermo Fisher, 62249, 1:2000) for 1 h. Samples were imaged using an Eclipse Ti2 spinning disk confocal microscope (Nikon) with a 60× oil objective.