St5010 autostainer xl

Tissues were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin. Formalin-fixed and paraffin-embedded sections were cut at a 3-μm thickness. Automated hematoxylin and eosin (H&E) staining was performed on a Leica (Wetzlar, Germany) ST5010 Autostainer XL instrument for all samples.

Biopsy specimens (approximately 1 cm³) were taken from ECRB tendon origins during surgery; they appeared dull, gray, friable, and edematous. Sampling was performed only after informed consent was obtained. Tendon degeneration in each harvested tissue was analyzed quantitatively. All specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4-μm serial sections for H&E and Alcian blue staining. H&E staining was performed using an automated staining machine (Leica ST5010 Autostainer XL). Alcian blue (pH 2.5) staining was performed on deparaffinized sections according to the standard protocols: Alcian blue staining at room temperature for 30 min followed by nuclear fast red staining for 3 min. The Movin score was adopted to classify degrees of tendinopathy based on H&E and Alcian blue staining [28 (link)]. The 4-point scoring system comprised 0 (normal), 1 (mild), 2 (moderate), and 3 (severe), by including the following assessment criteria: fiber structure, fiber arrangement, size and shape of cell nuclei, collagen stainability, extracellular matrix, vessel density, hyalinization of stroma, and proteoglycan content (Supplemental Table 1) [29 (link)]. All samples were blindly examined by an orthopedic surgeon, pathologist, and a medical student after proper training.

Formalin-fixed and paraffin-embedded tissues were sectioned at 4 μm. After paraffin sections are routinely deparaffinized to water, antigen retrieval was performed by high temperature ethylene diamine tetraacetic acid (EDTA) solution. All immunohistochemical staining procedures were performed on a Leica ST5010 Autostainer XL (Buffalo Grove, USA). 1:50 mouse anti-HMGA2 monoclonal antibody and horseradish peroxidase-conjugated mouse-anti-rabbit IgG were used as primary and secondary antibodies (Santa Cruz Biotech., USA); DAB was used for color development with hematoxylin counterstaining. The negative control is PBS instead of the primary antibody. HMGA2 protein is positive in brownish-yellow.

ApoE/LDLR^−/− mice were originally obtained from Jackson Laboratory (Sacramento, CA, USA; for reference, see: Ishibashi et al. 1994 (link)) and bred in house. Animals were kept in colony cages, in a temperature-controlled environment (22–25 °C) with a 12h:12h light/dark cycle. They had free access to food (cholesterol-free, pelleted diet; Sniff M-Z Spezialdiäten GmbH, Soest, Germany) and water.
All procedures involving animals were conducted according to the Guidelines for Animal Care and Treatment of the European Union and they were approved by the First Local Ethics Commission for Animal Experiments in Kraków (declaration No. 51/2009).
Six-month-old (established atherosclerosis) apoE/LDLR^−/− mice were used in this study. The animals were euthanized intraperitoneally with 100 mg/kg ketamine/10 mg/kg xylazine. Brachiocephalic arteries were dissected out, fixed in 4% buffered paraformaldehyde (pH = 7.4) for at least 48 h, and then routinely processed and embedded in paraffin (tissue processor TP1020; Leica, Wetzlar, Germany). Cross sections were cut every 5 μm along the artery (starting from the proximal end) and mounted serially on silanized slides. Sections were stained and mounted automatically using Leica ST5010 Autostainer XL combined with Leica CV5030 Glass Coverslipper.

Tissues were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin. Formalin-fixed and paraffin-embedded sections were cut at a 3-μm thickness. Automated hematoxylin and eosin (H&E) staining was performed on a Leica (Wetzlar, Germany) ST5010 Autostainer XL instrument for all samples.