Immunohistochemistry was performed with mAB 1D3 and 7A9 as well as phosphorylation-independent polyclonal TDP-43 (ProteinTech Group, Chicago, IL, USA; dilution 1:2000) and polyclonal C-t TDP-43 raised against amino acids 394-414 of human TDP-43 [16 (link)].
Tissue was either formalin-fixed with fixation-times ranging from 1 day up to 4 years or ethanol-fixed (1 day) and paraffin-embedded. Antigen retrieval was performed by boiling the sections in 10 mmol/L citrate buffer (pH 6.0) in a microwave oven. Immunohistochemistry was performed using biotinylated secondary antibodies and the avidin-biotin complex detection system (Vector Laboratories, Burlingame, CA, USA) with 3,3′-diaminobenzidine as chromogen. Double-labeling immunofluorescence was performed using Alexa Fluor 488 and 594 conjugated secondary antibodies (anti-rat IgG and anti-rabbit IgG, Molecular Probes, Eugene, OR, USA). 4′-6-diamidino-2-phenylindol (DAPI) (Vector Laboratories, Burlingame, CA, USA) was used for nuclear counterstaining.
Tissue was either formalin-fixed with fixation-times ranging from 1 day up to 4 years or ethanol-fixed (1 day) and paraffin-embedded. Antigen retrieval was performed by boiling the sections in 10 mmol/L citrate buffer (pH 6.0) in a microwave oven. Immunohistochemistry was performed using biotinylated secondary antibodies and the avidin-biotin complex detection system (Vector Laboratories, Burlingame, CA, USA) with 3,3′-diaminobenzidine as chromogen. Double-labeling immunofluorescence was performed using Alexa Fluor 488 and 594 conjugated secondary antibodies (anti-rat IgG and anti-rabbit IgG, Molecular Probes, Eugene, OR, USA). 4′-6-diamidino-2-phenylindol (DAPI) (Vector Laboratories, Burlingame, CA, USA) was used for nuclear counterstaining.
