Opal reagents

Multiplex immunofluorescence was performed on small cerebellar sections from FFPE
tissue blocks with OPAL reagents from PerkinElmer as described in the
PerkinElmer Multiplex IHC manual. Primary antibodies used are summarized in
eTable 1 (links.lww.com/NXI/A726, method: IF). Pretreatment (heating) in a
Braun household vegetable cooking device was performed in AR9 antigen retrieval
buffer from PerkinElmer for 40 minutes before the first antibody and with AR6
antigen retrieval buffer from PerkinElmer for 30 minutes between each antibody
staining.

We performed tyramide signal amplification labeling with the Opal reagents (PerkinElmer, Waltham, Massachusetts) using the Opal 4‐color automation IHC method. The primary antibodies used were as follows; LOX‐1 (ab126538, 1:800, Abcam, Cambridge, UK), CD15 (Carb‐3, 1:400, Dako, Glostrup, Denmark), CD11b (ab52478, 1:400, Abcam, Cambridge, UK), CD45 (1:400, #13917, Cell Signaling Technology, Inc., Danvers, Massachusetts), CD3 (ab16669, 1:300, Abcam, Cambridge, UK), CD20 (L26, 1:400, Dako, Glostrup, Denmark), CD163 (1:1000, #93498, Cell Signaling Technology, Inc., Danvers, Massachusetts), and α‐SMA (M0851, 1:100, Dako, Glostrup, Denmark). Antigens were retrieved from the tissue sections using ImmunoSaver (Nisshin EM, Tokyo, Japan). Tissue sections were incubated with fluorophores Opal 520, 570, and 690 for 10 minutes at room temperature. Antigen retrieval was performed using 10 mM sodium citrate buffer pH 6 in a microwave for 15 minutes. Finally, all sections were counterstained with DAPI. Images were captured using the BZ‐X700 microscope (Keyence). We analyzed 10 cases from the stromal LOX‐1 high group using triple‐labeled high‐power fields.