Immunohistochemistry of GSK3 in Human and Rat Tissue

Immunohistochemistry was performed on human control (temporal lobe) and rat tissue sections (40 μm thick) following established protocols (Kanaan et al., 2016 (link)). The tissue was incubated with purified GSK3 antibodies (12B2 – 1:500 or 15C2 – 1:1,000) overnight at 4° C, followed by goat anti-mouse biotinylated secondary antibody at 1:500 (115-065-166, Jackson Immuno Research) and then ABC Elite solution (according to the manufacturer’s instructions; PK-6100, Vector Labs, Burlingame, CA, USA). The tissue was developed using 3,3′-diaminobenzidine (D5637, Sigma) at 0.5 mg/ml in TBS-Tx with 0.003% H₂O₂ for 8 min. Control sections that were stained following the same procedure, but without the primary antibodies were performed (Supplementary Figure S3B). Images were acquired as z-stacks (0.9 μm step size) with a Nikon Eclipse 90i microscope, a Nikon DS-Ri1 camera, and Nikon Elements AR software (Nikon Instruments Inc., Melville, NY, USA), and the images (displayed using the extended depth of focus function) were prepared for publication using Adobe Photoshop and Illustrator.