Mouse anti spcas9

Cell cultures cultured on 8 well permanox chamber slides were permeabilized with 0.1% Triton X-100 in 0.1M PB for 10 minutes. Following this, the cells were incubated in a blocking solution consisting of 0.3% BSAc (Aurion), 0.05% sodium azide in 0.1 M PB for one hour. Subsequently, the cells were incubated in primary antibody (1:50 mouse-anti-SpCas9, Abcam; 1:150 rabbit-anti-Olig2, Millipore; or 1:200 chicken-anti-Olig2, AvesLab) in blocking solution overnight at 4 ºC. The samples were rinsed in 0.1 M PB and then incubated in secondary antibody blocking solution consisting of 0.5% BSAc (Aurion), 0.025% CWFS gelatin (Aurion), 0.05% sodium azide in 0.1M PB for 1 h, followed by incubation in secondary antibody (1:50 goat-anti-mouse IgG gold ultrasmall, Aurion) diluted in the same solution overnight at 4 ºC. To enhance gold labelling, we performed silver enhancement (R-GENT SE-LM, Aurion) for 15 to 25 minutes in the dark, followed by gentle washing in 2% sodium acetate and incubation in gold toning solution (0.05% gold chloride in water) for 10 minutes. The samples were then washed twice with 0.3% sodium thiosulfate in water. Finally we post-fixed with 2% glutaraldehyde (Electron Microscopy Sciences) in 0.1M PB for 30 minutes. Samples were rinsed and kept in 0.1M PB containing 0.05% sodium azide at 4 ºC until resin embedding.

Cells and sections were incubated in 1:200 Immunosaver (Electron Microscopy Sciences) in water at 60 ºC for 30 minutes. Peroxidase blocking was performed using a solution of 10% methanol and 10% H2O2 in 0.1 M PB. For permeabilization, the samples were washed 3 times for 5 minutes in PTA solution: 0.1% Triton X-100, 1 mg/mL bovine serum albumin (BSA) in 0.1 M phosphate buffer saline (PBS). The samples were then incubated in blocking solution (10% Casein, 5% normal goat serum in PTA) for one hour at room temperature. Subsequently, the samples were incubated overnight in primary antibodies diluted in blocking solution (1:300 rabbit-anti-Olig2, Chemikon; 1:100 mouse-anti-SpCas9, Abcam; or 1:1000 rabbit-anti-GFAP for cell culture, DAKO). The following day samples were thoroughly washed in PTA, and incubated in secondary antibody solution (1:500 AlexaFluor 488 goat-anti-mouse, Invitrogen; or 1:500 AlexaFluor 555 goat-anti-rabbit, Invitrogen). Samples were then washed in 0.1M PB and incubated in 1:1000 DAPI in water for 5 minutes. Finally, the samples were mounted with FluorSave (Calbiochem-Millipore).