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.
Brenner D., Sieverding K., Srinidhi J., Zellner S., Secker C., Yilmaz R., Dyckow J., Amr S., Ponomarenko A., Tunaboylu E., Douahem Y., Schlag J.S., Rodríguez Martínez L., Kislinger G., Niemann C., Nalbach K., Ruf W.P., Uhl J., Hollenbeck J., Schirmer L., Catanese A., Lobsiger C.S., Danzer K.M., Yilmazer-Hanke D., Münch C., Koch P., Freischmidt A., Fetting M., Behrends C., Parlato R, & Weishaupt J.H. (2024). A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice. The Journal of Experimental Medicine, 221(5), e20221190.
Other organizations :
Ludwig-Maximilians-Universität München, Munich Cluster for Systems Neurology, Charité - Universitätsmedizin Berlin, Freie Universität Berlin, Max Delbrück Center, Humboldt-Universität zu Berlin, Goethe University Frankfurt, German Center for Neurodegenerative Diseases, Inserm, Institut du Cerveau, Sorbonne Université, Assistance Publique – Hôpitaux de Paris, Central Institute of Mental Health, University of Mannheim, German Cancer Research Center
Morphological characteristics of mouse spinal cords
control variables
Fixation in 2.5% glutaraldehyde and 4% PFA in 0.1 M sodium cacodylate buffer at pH 7.4
Vibratome sectioning and immersion fixation in the same buffer for 24 h at 4°C
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)
En bloc uranyl acetate (1% aqueous uranyl acetate) contrasting
Graded dehydration in ethanol
Embedding in epon resin
Ultrathin sectioning and contrasting with 1% uranyl acetate and lead citrate
Semithin section contrasting with an equimolar mixture containing 1% methylene blue and 1% azure Blue
Annotations
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