Focal cerebral ischemia was generated by occluding the bilateral CCA for 30 min combined with permanent occlusion of the left distal middle cerebral artery (MCA) above the rhinal fissure, as previously described [2], [4], [16].
An alkaline phosphatase polymer was applied for 30 min, followed by staining with Permanent Red.
Adsorption was conducted in shaking flasks (50 mL biosuspension) for 10 min, and magnetic separation performed for another 10 min with a NdFeB block permanent magnet (B r = 1.3 T on the surface; Webcraft, Switzerland) according to the procedure described by Käppler [ 11].
For each sample, cryostat sections of 8 um were stained with hematoxylin for 10 min and eosin for 2 min, dehydrated in graded alcohol, and cover-slipped with permanent mounting solution after xylene clearing.
Rhesus iPSCs-derived cardiomyocytes (5 × 105/20 ul) were injected into the myocardium at the border zone of the infarct area with a 29-gauge needle 5 min after permanent LAD ligation.
Then, the slides were stained with Giemsa solution (5%) for 5 min and made permanent with EUKITT (Panreac).
MagnaBind magnetic beads (Pierce, Thermo Fisher Scientific, Bonn, Germany), pre-coated with anti-rabbit IgG antibody, were incubated with anti-irisin antibody (Adipogen) for 60 min under permanent agitation (Dynabeads MX Mixer, Life Technologies, Darmstadt, Germany).
A classical creep test with permanent loading during one week and a repeated minute-long loading test for which every 30 min, a loading is applied and kept constant during 5 min and finally totally removed.
They are shrubby, with permanent woody stems.
When MMI-0100 peptide was given 30 min after permanent LAD coronary artery ligation, the resulting fibrosis was reduced/prevented ~ 50% at a 2 week time point, with a corresponding improvement in cardiac function and decrease in left ventricular dilation.
