Picric acid solution

Collagen production was assessed as described by Remoué et al. (2013) (link). Briefly, HFF-1 cells were seeded and incubated as described previously for the cytotoxicity assay. After the 72 h incubation with peptides, cells were washed with PBS and the Sirius Red dye in picric acid solution (Sigma-Aldrich, St. Louis, MO, United States) was applied to each well. Cells were incubated at 25°C for 1 h under orbital shaking. Dye was then rejected, and cells were washed twice with absolute ethanol (AGA). Once the wells were dry, 1 M aqueous NaOH (Sigma-Aldrich, St. Louis, MO, United States) was added, and absorbance read at 540 nm (Biotek PowerWave XS).

Anhydrous solvents were either purchased as ultra-dry solvent from Acros Organics^® (Berlin, Germany) or received from a solvent purification system. Glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE) (85%, TCI Europe, Eschborn, Germany) were dried distilled, and stored over molecular sieves (5 Å). Dry toluene was obtained from MBRAUN SPS 800 solvent purification system (MBRAUN, Garching, Germany). Triethylamine (TEA, 99% Acros, Geel, Belgium), dry dimethylformamide (DMF, 99.8% Acros, Berlin, Germany), Iron (III) chloride hexahydrate (FeCl₃·6H₂O, 99% Grüssing, Filsum, Germany), Iron (II) chloride tetrahydrate (FeCl₂·4H₂O, 99% Grüssing, Berlin, Germany), ammonium hidroxide (NH₄OH, 25% Roth, Brooklyn, NY, USA), nitric acid (HNO₃, ≥65% Roth), picric acid solution (1.3% in water, Sigma Aldrich, Saint Louis, MO, USA), dry acetone (99% Acros, Berlin, Germany), (3-aminopropyl) triethoxysilane (APTES, 99% Sigma Aldrich), (1R, 8S, 9S)-bicyclo[6.1.0]non-4-yn-9-yl-methyl (4-nitrophenyl) carbonate (BCN-PNP), dansyl chloride (≥99% Sigma Aldrich), transferrin human (Sigma Aldrich), azido-dPEGTM12-NHS ester (Creosalus, Louisville, KY, USA), azido-dPEGTM8-NHS ester (Creosalus), azido-dPEGTM4-NHS ester (Creosalus) were used as received.

Kidneys were fixed in 4% formalin followed by paraffin embedding. 4 μm sections were sectioned and stained with Massons trichrome using standard protocols and the following reagents: Weigerts iron hematoxylin I and II (RH pharmacy, DK), Picric acid solution (Sigma), Briebrich Scarlet solution (Difco laboratories), wolframphoshporic acid hydrate (VWR) and methylene blue (Merck). The image analyses software Visiopharm (Visiopharm, Denmark) was used to measure glomerular diameter and determine kidney fibrosis. The glomerular diameter was defined as the widest part of the glomerulus and at least 25 glomeruli were measured per animal. Fibrosis was quantified automatically by Visiopharm as the blue stained area in Masson’s trichrome stained sections in the kidney cortex.