C dsd230

Femoral head tissues harvested from human patients, glucocorticoid-induced GONFH rats, Ctnnb1^Col2ER mice, and Ctnnb1^Sp7ER mice were viewed under a stereomicroscope (model C-DSD230, Nikon, Japan) to assess their appearance characteristics. The fresh femoral head tissues were placed on the test platform of a biomechanical testing machine (EnduraTec TestBench system, Minnetonka, MN). The axial compression load was applied at a speed of 0.5 mm/min until femoral head deformation. The loading-bearing stiffness of femoral head was calculated by testing software.

Mice were pretreated in different ways and then subjected to 30 min of ischemia, followed by 24 h of reperfusion. Then infarct size was evaluated using Evans blue and triphenyltetrazolium chloride staining (TTC) (Sigma-Aldrich), as described43 . Briefly, the left anterior descending coronary artery was ligated at 24 h after reperfusion, followed by retrograde injection of 2% Evans blue into the aortic arch. The heart was sliced into five sections of equal thickness perpendicular to the long axis, incubated in 1% TTC for 15 min at 37 °C and fixed in 10% formalin overnight. Images were captured using a microscope (DFC500, Leica, Germany) equipped with a digital camera (C-DSD230, Nikon, Japan). The left ventricle (LV) area, area at risk (AAR) and infarct area (IA) were determined using ImageJ (U.S. National Institutes of Health) and adjusted for weight. An example with Evans blue and TTC staining showing the delineation of infarct boundaries was shown in Supplementary Figure 1. AAR/LV% and IA/AAR% were calculated using the following formulas:


where W_n represents the weight of each heart section; W_T, the weight of the entire heart; I_n, percentage of infarct area (IA) with white color on each section; and A_n, the percentage of area at risk (AAR) with white and red color on each section.

Infarct size was evaluated using Evans blue and 2, 3, 5-triphenyltetrazolium chloride staining (TTC) (Sigma-Aldrich, St. Louis, USA) as previously described [18 (link)]. Briefly, rats were anesthetized after 120 min of reperfusion with the LAD re-occluded. We then injected 1 ml of 2% Evans blue via the aortic arch. The heart was harvested and sliced transversely into sequential 2 mm-thick sections, incubated in 1% TTC at 37°C for 30 min, and fixed in 10% formalin overnight. The left ventricular (LV) area, area at risk (AAR), and infarct area (IA) were determined using ImageJ (National Institutes of Health, USA) and adjusted for weight. Images were captured using a microscope (DFC500, Leica, Germany) equipped with a digital camera (C-DSD230, Nikon, Japan). AAR/LV and IA/AAR were calculated using the following formulas:
$\frac{\text{IA}}{\text{AAR}}=\frac{W_1\times I_1+W_2\times I_2+W_3\times I_3+W_4\times I_4+W_5\times I_5}{W_1\times A_1+W_2\times A_2+W_3\times A_3+W_4\times A_4+W_5\times A_5}$
$\frac{\text{AAR}}{\text{LV}}=\frac{W_1\times A_1+W_2\times A_2+W_3\times A_3+W_4\times A_4+W_5\times A_5}{W_T}$
where W_n represents the weight of each heart section; W_T, the weight of the entire left ventricle; In, percentage of infarct area (IA) (white areas) on each section; and A_n, the percentage of area at risk (AAR) (white and red areas) on each section. IA/AAR indicates the severity of myocardial injury while a consistent AAR/LV means the stability of the myocardial I/R model.