Bull s eye decloaker

Paraffin-embedded colon tissue were sectioned at 5 μm, deparaffinized and permeabilized with Triton X-100 (Sigma-Aldrich) and heat antigen retrieved with Bull’s Eye Decloaker (Biocare Medical). Slides were then blocked for endogenous peroxidase with Bloxall^™ blocking solution (Vector Lab) followed by blocking with either 3% bovine serum albumin, or mouse on mouse blocking reagent (Vector Lab). Anti-IL11 (ab10558; PA5-36544, Invitrogen), anti-CD45 (1:100; ab10558, Abcam), anti-LGALS3 (1 μg/ml; CL8942AP, Cedarlane) amd anti-LAMP2 (1 μg/ml; 550292, BD Bioscience) were added and incubated overnight at 4°C. Anti-rabbit (1:100; ab27478, Abcam) and anti-rat IgG (1 μg/ml; sc-2026, Santa Cruz) isotype controls were added as respective negative controls. Slides were incubated with anti-rabbit IgG peroxidase (1:500, A0545, Sigma-Aldrich) and anti-rat IgG peroxidase (MP-7404, Vector Lab) followed by chromogen development with ImmPACT^® DAB peroxidase substrate kit (SK-4105, Vector Lab) according to manufacturer’s instructions. Lastly, Gill’s haematoxylin (H-3401, Vector Lab) was added for nuclear counterstain. To control for unspecific binding, primary antibody isotype controls were included and images are presented in S3 Fig.

Paraffin-embedded slices were then rehydrated by incubation in a descending ethanol series and stored in Tris/HCl buffer pH 7.6. Slices were set into a container filled with Bull’s Eye Decloaker (1:20) in the autoclave (Biocare Medical). Conditions were as follows: 125°C for 30 s and 90°C for 10 s. Slides were subsequently cooled down and washed in Tris/HCl pH7.6. For background reduction, slices were incubated with 100% methanol, 30% H₂O₂ and Tris/HCl pH 7.6 in the proportion 1:1:8 for 30 min in a wet chamber. Afterwards, they were washed in Tris/HCl pH 7.6 and blocked in 2.5 μl Triton-X-100, 18.2 mg DL-Lysine and 998 μl 5% Tris-BSA for 30 min. Incubation with the first antibodies (anti-ATXN2, BD Biosciences, 611378, 1:50 and anti-FBXW8, Prestige, HPA038851, 1:20) took place overnight. After another washing step, slices were incubated with the secondary fluorescently-labeled antibodies (Cy3 and Cy2, Dianova, 711-225-152 and 715-165-150, 1:1000) for six hours and mounted with DAKO fluorescent mounting medium. Microscopic pictures were taken with a Nikon confocal microscope Eclipse 90i and a 60x magnification.