Immunohistochemical Analysis of Spinal Cord GAD67

A blocking solution (containing 5% milk, 1% bovine serum albumin, and 0.03% Triton X-100 diluted in 1× PBS) was applied to the previously sectioned spinal cords. Anti-GAD67 primary mouse antibody (Cat#: MAB5406; 1:500 concentration in blocking buffer) and anti-NEUN antibody (Cat#: ABN78; 1:500 concentration in blocking buffer) were applied overnight at 4 °C (Supplementary Table S1). The fluorophore-conjugated secondary antibodies (Cat#: A-21235 and A-11034; 1:500 concentration in blocking buffer) were applied for 90 min at room temperature (Supplementary Table S1). A 1× PBS solution was used to wash the cords three times during the staining process to minimize noise. The slides were imaged using a Nikon C2+ confocal laser microscope. The GAD67 images were quantified by image thresholding in Fiji, and the % area of positive GAD67 staining was recorded [50 (link)]. All images were analyzed in Fiji by a blinded examiner.