Avidin biotin complex detection system

Immunohistochemistry was performed on paraffin-embedded spinal cord tissues from individual IV-5 of family ALS53 (with R521C mutation) and another control individual without a neurological disorder. Patient tissue was kindly provided by P Blumbergs, J Manavis, and the NSW Brain Banks. Five-micrometre spinal cord sections were deparafiinised with xylene and rehydrated with a descending series of diluted ethanol and water. Antigens were retrieved by heating sections in 10 mM citrate buffer pH 6.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. Non-specific background was blocked with either 1.5% goat serum (Vector laboratories, Burlingame, CA, USA) for immunohistochemistry, or 1% bovine serum albumin (Sigma) for immunofluorescence. Sections were incubated in primary antibody, anti-LC3, at room temperature for 1 h. Alexa Fluor 488 (Life Technologies) was used as a secondary antibody. Biotinylated goat anti-rabbit IgG was used as secondary antibody for immunohistochemical staining. The avidin-biotin complex detection system (Vector Laboratories) with 3,3-diaminobenzidine as chromogen was used to detect the immunoreactive signal. The slides were counterstained with hematoxylin and a cover-slipped using DPX.

Sections were stained with anti‐LRG (IBL, Gunma, Japan) and anti α‐SMA (abcam, Cambridge, UK) antibodies. Primary antibodies were visualized using an Avidin‐Biotin Complex detection system (Vector Laboratories Inc., Burlingame, CA, USA).