Dako envision flex target retrieval solution

FFPE tissues were cut into 4.5-µm-thick sections using a microtome and immuno-stained with the monoclonal 5G4 anti-mouse antibody [44 (link)], which labels only the disease-associated αSyn (1:4000; 5 min pre-treatment with 80% formic acid; Roboscreen, Leipzig, Germany). Target retrieval was performed using the DAKO EnVision FLEX Target Retrieval Solution. The DAKO EnVision detection kit, EnVision FLEX peroxidase-blocking solution, 3,3′-Diaminobenzidine chromogen, and EnVision FLEX+ mouse linker (Dako, Glostrup, Denmark) was used to visualize the antibody reactions.
Digital images were obtained with TissueScope^TM LE120 and TissueSnap^TM (Huron, Saint Jacobs, ON, Canada) and cropped using HuronViewer software (Figure S1).

Immunohistochemical staining was performed as described previously [8 (link), 10 (link), 15 (link)]. Briefly, specimens were fixed with formalin and embedded in paraffin, and then sliced sequentially at a thickness of 3 μm. Samples were deparaffinized with xylene and rehydrated with graded alcohol. After rinsing with Tris-buffered saline, samples were processed for antigen retrieval with Dako EnVision FLEX Target Retrieval Solution (pH 9.0), using Dako PT Link at 97 °C for 20 min according to the manufacturer’s instructions. Samples were incubated with antibodies against EPB41L5 (1:1,000) for 30 min, and the Dako Envision FLEX system was used for visualization. The coloring reaction was performed with 3,3′-Diaminobenzidine (Dojin) for 5 min. Hematoxylin was used as a counterstain. The scoring of immunohistochemical samples was performed as described previously [15 (link)] by two different investigators, including one pathologist (K.C.H). The H-score value 33% from the top was chosen as the cutoff point for the high-expression group.