Dako pre treatment module

PUM spheroids were removed from the ULA plates using a cut 200-µl pipette tip at various time points and fixed in 10% neutral buffered formalin for 15 minutes. Spheroids were then suspended in 2% agar before being processed using the Bayer Tissue-Tek VIP E300 tissue processor (Bayer AG, Leverkusen, Germany). Processed spheroids were subsequently embedded in paraffin blocks and sectioned at 4 µm onto X-tra adhesive slides (Leica Biosystems, Wetzlar, Germany), for immunohistochemical (IHC) staining.
IHC staining was performed as previously described¹⁸ (link) using the Dako Pre-Treatment Module and the Dako Envision FLEX kit (Agilent Technologies, Santa Clara, CA), according to the manufacturer's instructions. Details of antibodies, antigen retrieval, and concentrations are provided in Table 1. Positive staining was visualized with an AEC substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Sections were counterstained with Mayer's hematoxylin (VWR, Leighton Buzzard, UK), dyed blue with Scott's tap water (Leica), and mounted using Aquatex aqueous mounting medium (Sigma-Aldrich). Slides were scanned using the Leica Aperio CS2 slide scanner at 20× magnification.