Allprotect tissue reagent

At the time of harvest, animals were anesthetized with a mixture of isoflurane and oxygen. Body weights were recorded at this time. Thoracic cavities were opened, and blood was collected with 20-gauge butterfly catheter via direct cardiac puncture. After blood collection, anesthetized animals were immediately transferred to a carbon dioxide chamber for euthanasia. Hind limbs were removed at the coxofemoral joint. The left limb was placed into 10% neutral buffered formalin for 48 h and then transferred to PBS for microCT analysis. After microCT imaging was complete, tibial length was measured using calipers. Limbs were then transferred to a 12.5% solution of ethylenediaminetetraacetic acid (EDTA) at pH 7 for decalcification. EDTA was replaced twice weekly for 6 weeks.
After the right hind limb was removed, the knee joint was exposed by dissecting through the quadriceps muscles and reflecting the patella distally. The infrapatellar fat pad (IFP) was removed from the patellar tendon, weighed, and then placed in All Protect Tissue Reagent (Qiagen) for gene expression analysis. Fat from the left epididymis (hence forth referred to as gonad fat) was removed and weighed. A section of masseter muscle was collected from each animal, and its width was measured.

Following sternotomy, hearts were washed in ice-cold buffered saline solution to remove blood residue. Biopsies were obtained from the infarct core (center of the scar on the left ventricle), remote myocardium (non-infarcted interventricular septum), and control myocardium (healthy animals). To ensure RNA stabilization, biopsies were preserved in Allprotect Tissue Reagent (Qiagen, Hilden, Germany) at room temperature. Total RNA was isolated using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany). RNA purity and integrity were assessed by spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and nanoelectrophoresis (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA).

Tissue material collected during surgery was secured for molecular analyses in Allprotect Tissue Reagent (Qiagen, Wroclaw, Poland, Cat No./ID: 76405) in an Eppendorf tube and stored at -20°C until molecular analyses.
Blood samples were collected from the vein of the ulnar fossa from women in the study and control groups; the samples were collected into tubes designated for clotting, after which the samples were centrifuged for 10 minutes, at 1,500 ×g, at 20°C to obtain serum for further biochemical analyses. The samples were stored at -20°C until the start of the analysis.

Ten obese nondiabetic male Zucker rats (OB) (fa/fa, 10 weeks of age) and 10 lean littermates (L) (Fa/?) were purchased from Charles River Laboratories (Calco, Lecco, Italy). The rats were housed at constant room temperature (22 ± 2°C) and humidity (60 ± 5%) with a light-dark cycle of 12 hours each and fed a standard rodent chow (10% fat) and water ad libitum. At the age of 25 weeks, the rats were anesthetized with zoletil (20 mg/kg) and sacrificed by cervical dislocation. Ten hearts (five L and five OB) were stored in Allprotect Tissue Reagent (QIAGEN, Hilden, Germany) at -20°C until RNA and protein extraction. The remaining hearts were fresh frozen in OCT for cryosectioning. Blood was obtained by cardiac puncture, and after cloating, serum was isolated by centrifugation at 1500 g for 15 min. The Italian Ministry of Health approved the procedures of animal care, anesthesia, euthanasia, and tissue collections for this study (Ministerial Authorization 325/2015PR of 2015/04/05).

Samples of visceral (around the omental fold and the liver), subcutaneous and intermuscular (between the thigh muscles) adipose tissue, the abdominal fat body and the liver were taken from 10 juvenile (4 years old) Nile crocodile at Izinthaba Crocodile Farm, Pretoria, South Africa (Latitude 25° 38' 46.00" S and Longitude 27° 57' 43.59" E). Pansteatitis samples of the visceral and intermuscular adipose tissues were collected from five adult (about 15 years old) Nile crocodiles, which were dying of pansteatitis in the wild at the Loskop Dam, Olifant River in Mpumalanga, South Africa (Latitude -25° 24' 6.59" S and Longitude 29° 16' 28.20" E. A total of 50 samples of normal healthy adipose tissue, (five samples per animal) and 10 pansteatitis samples consisting of five of each fat samples (pansteatitic visceral and intermuscular at 0.25 g each) were collected in duplicates into separate tubes of 2 ml Qiagen All Protect tissue reagent to prevent RNA degradation. The samples were incubated overnight at 4 ^oC before storage at—20°C and final transfer to—80 ^oC.