Normal buffered formalin

Epididymal fat pad samples were fixed for 48 to 72 hours at RT with 10% Normal Buffered Formalin (Sigma, cat.:HT501128-4L). The specimens were then dehydrated in 50% EtOH to 70% EtOH gradient and kept at 4°C in 70% EtOH until paraffin embedding at Histology core facility of the Institute of Biomedicine, University of Turku, Finland. Four µm sections were cut, dried overnight at 37°C, deparaffinized, rehydrated, stained with Periodic Acid Schiff (Sigma-Aldrich, cat.:395B-1KT), and counterstained with hematoxylin. Slides were mounted with Dibutylphthalate Polystyrene Xylene (DPX; Sigma, cat.:06522-100ML). Stained sections were imaged using Pannoramic 1000 (3DHISTECH) scanner (with a 40x lens) and analyzed using CaseViewer software (3DHISTECH – CaseCenter 2.9 SP1).

Animals were sacrificed three weeks after lubricin injection (four weeks after DMM surgery) and intact knee joints were dissected and fixed in 4% normal buffered formalin (Sigma, St. Louis, MO). Samples were decalcified and embedded in paraffin (VWR, Radnor, PA). Ten μm thick, longitudinal serial sections were stained with Safranin O (Fisher, Waltham, MA) to visualize proteoglycans. Whole joint sections, medial, lateral and the ACL/PCL insertion sites were graded for signs of OA according to the OARSI Guidelines for rat knee joints34 (link). Sections were deparaffinized in CitraSolv (Fisher Scientific; Fairlawn, NJ) and rehydrated through a series of graded ethanol to distilled water steps. Antigen retrieval (10 mM sodium citrate, pH 6.0, Fisher Scientific) and blocking (1:500 dilution; 100 μL rat serum:50 mL TRIS-buffered saline, 0.1% Tween 20 (TBST) for 1 hr), steps were performed prior to going through sequential wash (TBST) and primary antibody application steps. A primary antibody for NFkB P65 antibody (Cell Signaling Technology), or lubricin12 (link) bound to Alexa 488 (Molecular Probes) and the nucleic acid stain DAPI (Sigma) were applied to sections. After antibody staining, sections were mounted using FluorSave reagent (Calbiochem) and coverslipped.

Whole femurs were dissected from mice and immediately fixed in 10% normal buffered formalin (Sigma Aldrich) for 72 hours at room temperature. The bones were then decalcified for 21 days at 4 °C in 14% tetra-EDTA (Affymetrix), the pH of which was adjusted to 7.4 using glacial acetic acid. The decalcification solution was changed three times a week during this period.
The tissue was then embedded in paraffin using a standardised automated process at our institution following which, 4 μm-thick longitudinal sections were cut and mounted on SuperFrost glass slides; these were allowed to air dry for a week.
Frozen, calcified mouse femurs from the same animals were used for COX/SDH histochemistry. Whole femurs were embedded in OCT and 6 μm-thick sections were cut using a Leica Cryojane tape transfer system. The tissue sections were left to air-dry for one hour prior to performing COX/SDH histochemistry.