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 pre-incubated in a 50 mM Trizma buffer containing 1.2 mM MgCl2 and 2.0 mM CaCl2 at room temperature for 20 min. After [11C]MMPIP (74 MBq, 0.7 nmol) was added to the incubation buffer (0.2 L), the brain sections were incubated in the buffer for 60 min 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.
Twenty-micrometer-thick sagittal rat and monkey brain sections were incubated in an autoradiography buffer containing 5 nM [11C]TASP0410457 as described in Additional file 1: Supplemental methods.
Briefly, the brain sections were incubated in 0.5 MBq/ml [ 18 F]flutemetamol in 2% human serum albumin for 30 min.
For immunohistochemical staining of brain sections or fixed organotypic brain slices, sections were incubated with a combination of mouse anti-Tuj1 antibody, C-Ter rabbit antibody, and chicken anti-GFP antibody (each diluted at 1 1000) overnight at room temperature in PBS (0.02 M) containing 0.3% Triton and 0.02% sodium azide (PBS-T-azide).
Briefly, brain tissue sections were incubated in freshly prepared permeabilization solution (0.1% (w/v) Triton X-100, 0.1% (w/v) sodium citrate) for 2 min on ice.
For fluorescent immunohisochemistry, 6 µm paraffin embedded sections were cut on a microtome, and brain and spinal cord sections were incubated in phosphate buffered saline (PBS, pH 7.4) with 5% normal goat serum (NGS) and 0.1% TX for 1 hr at RT.
Briefly, all brains were serially sectioned, and 8- μm sections were incubated with mAb anti-human-specific MHC class-I IgG (10 μg ml 1, 2 h, at 4°C; mAb W6/32, Serotec, Inc ., followed by a multi-link secondary antibody conjugated to biotin and horseradish peroxidase, followed by incubation with streptavidin, and then 3,3′-diaminobenzidine substrate (Sigma, St Louis, MO, USA).
Sections were incubated overnight under light protected vacuum to allow an optimal fixation of brain sections on slides.
