BM was collected from femurs and tibias as described before (Ulland et al., 2017 (link)). To prepare BMM for antibody binding, survival, and immunoprecipitation assays, BM cells were counted and plated at 10 × 106 cells/100-mm Petri dish in RPMI supplemented with Glutamax, penicillin/streptomycin, nonessential amino acids, pyruvate, 10% heat-inactivated fetal bovine serum (complete RPMI) and 50 ng/ml CSF-1 (Peprotech). Cells were cultured for 7 d before use.
To perform scRNA-seq of the immune cells in brain, whole brains of 22-wk-old (5.5 mo) mice from AL002c- or control IgG-treated groups were dissected after transcardial perfusion with ice-cold PBS and dissociated as previously described (Song et al., 2018 (link)). Immune cells were recovered after Percoll (GE) separation, as described (Mildner et al., 2007 (link)).
To perform the biochemical analysis of brain after AL002c chronic treatment, brains were removed after transcardial perfusion with ice-cold PBS. Cortex and hippocampus were dissected out from left brain hemispheres and flash frozen; right brain hemispheres were fixed in 4% PFA for 2 d at 4°C and rinsed in PBS before incubation for 2 d at 4°C in 30% sucrose. Dehydrated brain tissues were frozen in a 2:1 mixture of 30% sucrose and optimal cutting temperature compound for obtaining serial 40-µm cryosections.
To perform scRNA-seq of the immune cells in brain, whole brains of 22-wk-old (5.5 mo) mice from AL002c- or control IgG-treated groups were dissected after transcardial perfusion with ice-cold PBS and dissociated as previously described (Song et al., 2018 (link)). Immune cells were recovered after Percoll (GE) separation, as described (Mildner et al., 2007 (link)).
To perform the biochemical analysis of brain after AL002c chronic treatment, brains were removed after transcardial perfusion with ice-cold PBS. Cortex and hippocampus were dissected out from left brain hemispheres and flash frozen; right brain hemispheres were fixed in 4% PFA for 2 d at 4°C and rinsed in PBS before incubation for 2 d at 4°C in 30% sucrose. Dehydrated brain tissues were frozen in a 2:1 mixture of 30% sucrose and optimal cutting temperature compound for obtaining serial 40-µm cryosections.
