Immunhistochemistry
NEFL
PATHOLOGY
Deparaffinization, rehydration and antigen retrieval of tissue sections were done using the PT Link instrument from Dako (Agilent, Santa Clara, CA, US) according to the manufacturers protocol. Sections were blocked in PBS containing 30% SEA BLOCK (Thermo Fisher, #37527) and 1% Triton X-100 (Sigma-Aldrich, #X100) for 90 minutes. Antibodies were diluted in PBS containing 10% SEA BLOCK and 0.1% Triton X-100 and incubated for 1 h. After each antibody incubation, the sections were washed 3x 5 minutes in PBS. Cover glasses were mounted using ProLong Glass with NucBlue (Thermo Fisher, #P36980). Antibodies used: MAC-3 (BD Biosciences, #550292; 1:100), NEFL (Thermo, #PA1-32240; 1:100), GFAP (Sigma, #HPA056030, 1:500), NOGO-A (Thermo Fisher, #PA5-20367; 1:100), human nuclei (Sigma-Aldrich, #MAB1281, 1:100), Alexa Fluor 647 goat anti-rabbit (Thermo Fisher, #A21245; 1:500) and Alexa Fluor 647 goat anti-rat (Thermo Fisher, A21247; 1:500). Luxol Fast Blue staining, used for myelin staining, was performed by the pathology department at Haukeland University Hospital (Bergen, Norway).