Ultra view dab brown

Mouse tibias/femurs and BM core biopsies from SLE patients and controls were fixed in 10% neutral buffered formalin for 1hr and decalcified in Rapid-Cal-Immuno-Decal Solution (BBC Biochemical, Stanwood, WA) for 2hr. Paraffin sections (4-µm) were stained with H&E. For IHC, paraffin sections were dried on slides for 2hr at 60°C. Slides were placed in a Ventana Medical Systems (Tucson, AZ) automated immunostainer and deparaffinized. Heat-induced epitope retrieval was performed with Ventana’s CC1 retrieval solution (30 min at 95–100°C). Primary antibodies anti-cleaved-caspase-3 (Cell Signaling, Danvers, MA), anti-TNFα (Abcam, Cambridge, MA), and anti-CD71 (Dako/Agilent Technologies, Santa Clara, CA) were applied for 32min at 37°C followed by peroxidase- or alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (30min). Reaction product was visualized using Ultra View DAB (brown) or Alkaline Phosphatase Red detection kit (Ventana). Slides were counterstained with Ventana hematoxylin.

BM core biopsies were fixed in 10% neutral buffered formalin and decalcified (6 (link)). Four-μm sections were deparaffinized and underwent heat-induced epitope retrieval before staining with anti-cleaved-caspase-3 (Cell Signaling), anti-TNFα (Abcam), and anti-CD68 (Dako) antibodies followed by peroxidase- or alkaline phosphatase-conjugated goat secondary antibodies (6 (link)). Reaction product was visualized using Ultra View DAB (brown) or Alkaline Phosphatase Red kits (Ventana). Slides were counterstained with hematoxylin. Numbers of activated caspase-3⁺ cells (red) that did not co-localize with macrophages (brown) were determined as the mean number of red⁺brown⁻ cells per 100X field (4 fields per patient).