Detection of seeded pathology using tyramide amplification

Mount the fixed floating 40-micron sections onto gelatin-coated slides and dry at Room temperature 0h 20m 0s.

Rehydrate the slides in TBST (refer materials section) and digest with proteinase K (PK, 20) diluted in TBST for 0h 20m 0sat 37°C.

Fix the slides in 4% paraformaldehyde for 0h 20m 0s, rinse 3 times in TBST, and incubate with 3% hydrogen peroxide for 0h 30m 0s to quench endogenous peroxidases.

Place the slides in blocking buffer (TBST, 3% bovine serum albumin, 2% goat serum) for 1h 0m 0s and then incubate it 1h 0m 0s at 4°C in blocking buffer containing anti-PSER129 antibody EP1536Y (Abcam) diluted 1:50,000.

Next day, wash the slides 3 times in TBST and incubate with biotinylated anti-rabbit antibody (Vector Labs) diluted 1:400 in blocking buffer for 1h 0m 0s.

Wash the slides 3 times in TBST and incubate with avidin-biotin complex (ABC) reagent (Vector labs) diluted in blocking buffer for 1h 0m 0s.

Wash the slides twice with borate buffer 0.1 Sodium tetraborate 8.5) and incubate in borate buffer containing 0.003% hydrogen peroxide and 5micromolar (µM) biotinyl tyramide (Sigma-Aldrich) for 0h 30m 0s.

Wash the slides 3 times in TBST and incubate with ABC reagent for 1h 0m 0s.

Heating the slides for 0h 30m 0s at 80°C in 20millimolar (mM) citrate buffer 6.0 before ABC reagent can increase detection sensitivity.

Wash the slides in TBST and develop using nickel-enhanced 3,3'-Diaminobenzidine DAB as previously described (refer references section).

Counterstain the slides with methylgreen (Sigma), dehydrate with graded alcohols, clear with xylenes, and cover the slides using cover slipps with cytoseal 60 (Fisher Scientific).

Perform the Brightfield microscopy using Nikon A1 laser scanning microscope.

Perform the density analysis including binary masks and region of interest (ROI) analyses using Elements software (Nikon).