Shandon zinc formal fixx

The atherosclerotic plaque material of 22 anonymous human subjects was collected peri-operatively from carotid (n = 10) and femoral (n = 12) artery endarterectomy (from July to December 2016 at the Haaglanden Medical Center Westeinde, The Hague, The Netherlands). The handling of all of the human samples complied with the “Code for Proper Secondary Use of Human Tissue”, METC number 16-071. The plaque samples were placed in RPMI (Lonza, Breda, The Netherlands) directly after removal from the patient. The culprit part of the plaques was collected as described previously [23 (link)], and stored in Shandon Zinc Formal-Fixx (Thermo Scientific, Waltham, MA, USA) for histology purposes. The remainder (~90%) of the plaques were processed into single cell suspensions by a 2-h digestion step in 37 °C, with an enzyme mix consisting of collagenase IV (Thermo Scientific, Waltham, MA, USA) and DNase (Sigma, Zwijndrecht, The Netherlands), as previously described [24 (link)]. Subsequently, the samples were filtered through a 70 μm cell strainer to obtain single cells, which were kept in RPMI/1% Fetal Calf Serum (FCS) until further analysis.

Hearts of the transplanted LDL receptor knockout recipients (obtained at sacrifice, i.e. after the 20 week WTD challenge) were fixed in 4% Shandon Zinc Formal-Fixx (Thermo Fisher Scientific, 9990245) for 24 h and subsequently embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, USA) until further processing. Cryosections (7 μm) at the level of the aortic sinus were obtained using a Leica CM3050s cryostat. Atherosclerotic plaques were stained with Oil Red O for neutral lipids and Masson’s Trichrome to identify collagen. Plaque area (in µm²) and lesional collagen content quantifications were performed blinded using a Leica image analysis system (Leica Ltd, Cambridge, UK).