Opal multiplex ihc kit

Eyes were enucleated and fixed in 4% paraformaldehyde (AR1068; Boster Bio, Pleasanton, CA, USA) for 48 hours at 4°C. Whole eyeballs were dehydrated and infiltrated with paraffin using a tissue processor (Tissue-Tek VIP 6; Sakura Finetek USA, Inc., Torrance, CA, USA) and subsequently embedded into paraffin blocks on a HistoCore Arcadia Embedding Center (Leica Biosystems, Melbourne, Australia). Three-micrometer sections were cut at the level of the optical nerve and hematoxylin and eosin and Masson's Trichrome staining was performed according to standard protocols. Immunofluorescence staining was carried out on the automated Leica Bond platform (Leica Biosystems) using the Opal Multiplex IHC Kit (Akoya Biosciences, Menlo Park, CA, USA). Antibodies used in this study and details about the antigen retrieval are listed in the Table. Nuclei were stained with spectral DAPI (FP1490; Akoya Biosciences) and slides mounted with ProLong Antifade Mounting Medium (P36961; Invitrogen, Carlsbad, CA, USA). Tissues were imaged using an Axio Scan.Z1 slide scanner (magnification ×20; Carl Zeiss Microscopy GmbH, Jena, Germany). Iba1⁺ and F4/80⁺ area was quantified using HALO image analysis software (Indica Labs, Albuquerque, NM, USA).