Masson s trichrome staining

At 1-month and 2-month timepoints, samples were excised from the subcutaneous tissue and fixed with 4% paraformaldehyde for 24 h. After fixation, samples were washed with increasing percentages of ethanol (70–100%) for 30 min each, washed thrice with xylene, and embedded in paraffin wax blocks for sectioning. Slices (10–30 µm thick) were cut using a Leica Biosystems microtome for histological analysis before being stained using hematoxylin and eosin stains or Masson’s Trichrome staining using protocols available through Sigma–Aldrich. Analysis was performed using light microscopy (Leica, ×4 and ×10 objectives) and image stitching was performed in ImageJ (NIH, Bethesda, MD). Brightfield images were analyzed and qualitatively assessed for general inflammation compared to PLLA control samples. Samples were also analyzed for a number of inflammatory cells utilizing a modified scoring system designed by the International Organization for Standardization (ISO 10993-6 Annex E). Scoring was based on a scale from 0 to 4 (0 = none; 1 = Rare, 1–5 Minimal; 2 = 5–10, Mild; 3 = Heavy Infiltrate, Moderate; 4 = Packed, Severe).

Masson’s trichrome staining (Sigma-Aldrich), and Sirius red staining (Abcam, Cambridge, UK) were performed to evaluate glomerular sclerosis. For the immunohistochemical assays, paraffin-embedded kidneys were cut into 4-μm-thick slices, deparaffinized, and hydrated using xylene and ethanol. Endogenous streptavidin activity was blocked using 3% hydrogen peroxide. The deparaffinized sections were stained with an anti-KLF2 antibody for kidneys and then incubated with horseradish-peroxidase-conjugated anti-mouse IgG (DAKO, Carpinteria, CA, USA, K3954). Next, 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) was used for immunohistochemical detection. Finally, all samples were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) and evaluated under a light microscope (DFC-295; Leica, Mannheim, Germany). For each sample, five fields (X400) were randomly selected, and blue- (Masson’s trichrome staining) as well as brown-stained areas (immunohistochemistry) that reflected kidney fibrosis were quantified using computer-based morphometric analysis (Qwin 3; Leica). Scoring was performed in a blinded manner using the mean values of the positive areas (%).