Azure blue

Mouse spinal cords were fixed in 2.5% glutaraldehyde and 4% PFA in 0.1 M sodium cacodylate buffer at pH 7.4 after deep anesthesia perfusion. Spinal cords were vibratome-sectioned and immersion fixed in the same buffer for 24 h at 4°C. After tissue trimming and washes in 0.1 M sodium cacodylate buffer, postfixation in reduced osmium (2% osmium, 2.5% potassium ferrocyanide in 0.1 M cacodylate buffer) was followed by en bloc uranyl acetate (1% aqueous uranyl acetate) contrasting, graded dehydration in ethanol, and embedding in epon resin (Serva). After ultrathin sectioning, the grids (UC7 Ultramicrotome; Leica) were contrasted by 1% uranyl acetate and lead citrate (Ultrostain; Leica). Semithin sections were contrasted by an equimolar mixture containing 1% methylene blue (Carl Roth GmbH & Co. Kg) in 100 ml sodium tetraborate and 1% (1 g) azure Blue (Carl Roth) in 100 ml water.

Freshly plasma-coated ACLAR (plastic) films (Science Services) were put into the cell culture dish before seeding of TBK1^wt/wt and TBK1^E696K/E696K MEFs. 5% glutaraldehyde in 0.2 M cacodylate buffer prewarmed to 37°C was added 1:1 to the cell culture medium and replaced by 2.5% glutaraldehyde in 0.1 M cacodylate buffer after 5 min. Dishes were incubated for a further 25 min on ice. Cells were washed 3 × 5 min with 0.1 M cacodylate buffer on ice and stored in buffer at 4°C until post-fixation. After washes in 0.1 M sodium cacodylate buffer, post-fixation in reduced osmium (2% osmium, 2.5% potassium ferrocyanide in 0.1 M cacodylate buffer) was followed by en bloc uranyl acetate (1% aqueous uranyl acetate) contrasting, graded dehydration in ethanol, and embedding in epon resin (Serva). After ultrathin sectioning, the grids (UC7 Ultramicrotome; Leica) were contrasted by 1% uranyl acetate and lead citrate (Ultrastain; Leica). Semithin sections were contrasted by an equimolar mixture containing 1% methylene blue (Carl Roth GmbH & Co. Kg) in 100 ml sodium tetraborate and 1% (1 g) azure Blue (Carl Roth) in 100 ml water.