Dx61 optical microscope

Immunohistochemical studies were performed in sections of paraffin-embedded joints from lean and db/db mice (mice with mutated receptor of leptin) obtained from a previous study of our group [36 (link)]. Samples were deparaffinised, cleared with xylene, and hydrated in a series of increasing grade alcohol. Heat-mediated antigen retrieval was performed in citrate buffer (pH 6.0; S2369; Dako, Agilent Technologies Spain S.L., Barcelona, Spain) for Nrf-2 detection or in ethylenediaminetetra-acetic acid (EDTA) buffer (pH 9.0; S2367; Dako) for CBS and CSE detection. Thereafter, peroxidase blocking solution (Dako) was used to block endogenous peroxidase activity. Then, slides were washed with phosphate buffer solution and incubated with primary antibodies against Nrf-2 (1:200; sc-13032; Santa Cruz Biotechnology), CBS (1:400; ab54883; Abcam), and CSE (1:400; ab54573; Abcam). Antigen–antibody interactions were determined with the rabbit/mouse peroxidase/DAB DAKO REAL EnVision Detection Kit (K5007; Dako). Sections were counterstained with haematoxylin and eosin. Slides were dehydrated in graded alcohol, cleared in xylene, and mounted in DePeX (Dako). Slides were visualised in an Olympus Dx61 optical microscope (Olympus España S.A.U., Barcelona, Spain). Staining intensity was quantified using ImageJ software.

Cartilage explants were sectioned at 4 μm thickness to show all layers of cartilage. Following slide de-waxing and rehydration, samples were stained with Toluidine blue (TB) (Merck, Darmstadt, Germany) dye. TB is a basic thiazine metachromatic dye with high affinity for acidic tissue components, and in cartilage, a purple color indicates a high amount of hyaluronic acid (HA), its most abundant metachromatic component.
All slides were then visualized using an Olympus Dx61 optical microscope (Olympus España S.A.U., Barcelona, Spain) and were subsequently imaged. The image quantification was developed using software ImageJ Fiji 1.54f [70 (link)].