Brain tissue sections incubated with scFvs were washed and incubated for 3 h with anti-His6 antibodies (0.3 ug/ml, Roche) at RT. Finally, all sections were washed and incubated for 1.5 h with anti-mouse HRP-labelled antibodies (diluted 1∶1000, Jackson ImmunoResearch) at RT.
Thus, the sections incubated with the first primary antibody and revealed with FITC-TSA were incubated with Cy3-conjugated donkey anti-rabbit antibody.
Negative controls included sections incubated with the secondary antibodies alone.
Briefly, after normal serum blocking and before primary antibody incubation, the mouse brain sections were incubated with unconjugated affiniPure Fab fragment goat Anti-Mouse IgG (H + L) (Jackson ImmunoResearch Labs, West Grove, PA) for 1 hr at room temperature.
Brain sections were incubated with AV-45 radiotracer and attached to the phosphor imaging plates for 24 hours.
Briefly, brain sections were incubated with the sea lamprey gabab1 DIG-labelled probe at 70 °C and treated with RNAse A (Invitrogen, Massachusetts, USA) in the post-hybridization washes.
Frozen brain sections were incubated with the primary antibody against synaptophysin (1∶100, 60min, RT, Chemicon, MAB5258), followed with Cy3 labeled secondary antibody.
Brain sections were incubated with anti-BrdU antibody and anti-NeuN antibody (to mark mature neurons) or anti-GFAP antibody to identify astrocytes.
For immunostaining, brain sections were incubated with mouse monoclonal antibody specific to Human nuclei (1∶100; MAB1281, Chemicon, Temecula, CA) to evaluate the migration of transplanted HMO6 cells.
