*4.2. Histology and Immunohistochemistry*

From the paraffin-embedded samples, 10 μm thick sections were obtained and stained with hematoxylin–eosin (HE) and Sirius red. For each case the IFP's score was applied considering the presence of lymphocytic infiltration and vascularization as published [21]. The adipose lobules dimension and the thickness of the interlobular septa were not evaluated due to the small volume of the IFP biopsies obtained from the ACL patients and gene expression analysis was not performed in all patients of this group.

All sections were analyzed under a DM4500-B light microscope (Leica Microsystems, Wetzlar, Germany) and recorded in full color (24 bit) with a digital camera (DFC 480, Leica Microsystems).

Collagen subtypes were studied on sections stained with Sirius red and quantified as previously described [41].

For immunohistochemical (IHC) analysis, an anti-IL-6 antibody (polyclonal mouse antibody, Thermo Fisher) was used at 1:200 without antigen retrieval. Tissue sections were incubated using the DAKO Autostainer System (EnVision™ FLEX, High pH). The presence of positive cells was evaluated and graded as follows: grade 0 = absence of positive cells, grade 1 = weak positive cells, grade 1 = rare strong positive cells, grade 2 = clustered strong positive cells, grade 3 = diffuse strong positive cells.
