Immunofluorescence Cryosectioning and Aggresome Detection

We performed immunofluorescence experiments on cryosections of 25 microns and proceeded as previously described (Terzibasi Tozzini et al., 2012 (link)). Briefly, we washed the sections in PBS to remove the cryoembedding medium. We then performed an acid antigen retrieval step (10 mM trisodium citrate dehydrate, 0.05% tween, at pH 6) and (when required) stained the section with the ProteoStat Aggresome Detection Kit (Enzo Life Sciences Inc.) as previously described (Shen et al., 2011 (link)). We applied a solution 1:2000 of Aggresome dye in PBS for 3 min, rinsed the samples with PBS, and left the sections immersed in 1% acetic acid for 40 min. We applied the blocking solution (5% BSA, 0.3% Triton‐X in PBS) for 2 h. Primary antibodies at proper dilution were added in 1% BSA, 0.1% triton in PBS, and left overnight at 4°C (Table S3). The day after, we applied secondary antibodies (1:400 dilution) in the same solution. After 2 h at room temperature, slides were washed three times with PBS and mounted with Fluoroshield DAPI mounting medium (Sigma‐Aldrich).
For all further quantifications, slides from sets of 4–5 animals per group (young and old) were used.