2.2. Histology

Explanted silicone pieces and surrounding capsules were fixed overnight in formaldehyde at 4°C and transitioned to 70% ethanol. Samples were then paraffin embedded and sectioned at 4 μm. Samples were stained with H&E (Figure 1E) or Picro‐Sirius Red. Immunohistochemistry sections were deparaffinized, rehydrated, and blocked for endogenous peroxidase activity (3% H₂O₂ in H₂O) and nonspecific background staining (3% normal goat serum in Background Sniper, BioCare Medical). Antigen retrieval was performed with citrate buffer (pH 6.0) or Tris‐EDTA (pH 9.0) in a pressure cooker for 10 min, and slides were incubated for 30 min at room temperature with primary antibodies. Primary antibodies (all from Abcam) included α‐SMA (ab124964), CD31 (ab28364), CD68 (ab125212), iNOS (ab15323), CD163 (ab182422), Ki67 (ab15580), ER‐α (ab271827), and ER‐β (ab3576). Primary antibody binding was detected by subsequent incubation with species‐appropriate biotinylated IgG (Vector Laboratories), followed by streptavidin‐horse radish peroxidase (Vector Laboratories) and chromogenic development with 3,3‐diaminobenzidine (Vector Laboratories). Tissue sections were counterstained with Gill’s hematoxylin (Vector Laboratories), slides were dehydrated, and a cover slip was placed. Slides were imaged on a Zeiss Axio Observer Z1 inverted microscope (Zeiss) and quantified using ImageJ (NIH).

Images were quantified by examining the area within the fibrous capsule on the side oriented with overlying skin at the midpoint of the implant to maintain consistency and remove potential variability from the dissection plane on the other side of the implant created at explan ation. Due to the substantial differences in total capsular area, cellular histological stains were quantified using two methodologies, both as a percent of total cells within the capsule to determine differences in the makeup of the capsular tissue and as cells/mm of the implant surface to determine differences in the total amount of each cell type present.