Declere buffer

Paraffin-embedded tissue sections were cut at 10-μm-thick. Immunohistochemical (IHC) began with microwave antigen retrieval for 6 min (power 8) using Trilogy buffer (Cell Marque; Rocklin, CA) for CD68 and Declere buffer (Cell Marque; Rocklin, CA) for IBA1. Sections were then placed in 3% H₂O₂ in methanol for 30 min. Following washes in distilled water, sections were blocked in 5% goat serum at room temperature for 1 h. Sections were incubated in primary antibodies IBA1 (rabbit polyclonal, 1:1,000 IHC, Wako); CD68 (clone KP1) (1:50 IHC, Dako) overnight at 4°C. A biotinylated secondary antibody (Vector Laboratories) was amplified using avidin-biotin substrate (ABC solution, Vector Laboratories catalog no. PK-6100), followed by color development in Nova Red (Vector Laboratories). Immunofluorescence (IF) staining was done following microwave antigen retrieval for 6 min (power 8) using Declere buffer (Cell Marque; Rocklin, CA) for primary antibodies to: IBA1 (rabbit polyclonal, 1:250 IF, Wako); and PHF-1 (1:500 IHC and IF, a kind gift from Dr Peter Davies, Bronx, NY), and visualized using appropriate secondary antibody conjugated to an Alexafluor probe (1:200, Lifetechnologies) applied for 1 h. A 0.1% solution of Sudan Black was used to reduce autofluorescence. Slides were coverslipped using Vectashield mounting medium with DAPI (Vector Labs, Burlingame, CA).