Oct embedding compound

Ovaries were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS, cryoprotected in 15% sucrose (Fisher Scientific), embedded in optimal cutting temperature (OCT) embedding compound (Electron Microscopy Sciences), and stored at −80°C until sectioning. Ovaries were serially sectioned at 7 µm thickness and slides were stored at −80°C until staining. Slides were immunostained using primary antibodies to phosphorylated H2A histone family, member X (γH2AX), 4-hydroxynonenal (4-HNE), p53 up-regulated modulator of apoptosis (PUMA), and secondary antibody as detailed in Table 1. Diaminobenzidine substrate (Roche) was used for visualization of antibody binding, and sections were counterstained with Gill's hematoxylin. Scoring was done blind to treatment group as described previously (Mishra, et al. 2016 (link)). The percentages of follicles with positive granulosa cells or positive oocytes per section were calculated, and the percentages from the six scored sections per ovary were averaged for analysis.

Our first step, in vivo, was to establish that EPCs from the construct had the ability to migrate from the Construct into the myocardium. In order to assess EPC migration, eGFP⁺ EPCs from transgenic rats were utilized to create the Construct (20 mg/ml Fibrin, 17 × 10⁶ EPCs/ml). This Construct was then implanted onto ischemic myocardium following LAD ligation. The hearts were explanted 1 week following implant, flushed with PBS, and distended with OCT embedding compound (Electron Microscopy Sciences). Visualization was performed in the peri-infarct borderzone, which was defined as one microscopic field from the infarct. Hearts treated with Construct were compared to IC. Sections were briefly washed, then fixed in 4% paraformaldehyde and then stained for anti-GFP and anti-α smooth muscle actin (SMA, pericytes). Primary antibodies used for indirect immunofluorescence were goat anti-GFP (1:200, Rockland) and rabbit anti-SMA (1:150, Abcam). Secondary antibodies included donkey anti-goat conjugated to FITC (1:200, Abcam), and donkey anti-rabbit conjugated to Alexa Fluor 594 (1:200, Abcam). Nuclei were counterstained with DAPI (Vector Laboratories). Construct was compared to Control (isolated LAD ligation) and IC.

Adult (4-month-old) female Sprague–Dawley rats (Charles River Laboratories, Boston, MA; RRID:RGD_734476) were anesthetized with 4% isoflurane and oxygen and maintained at this level of anesthesia via nose cone in preparation for thoracotomy. The chest was then opened and the heart rapidly exposed. A needle was inserted into the left ventricle, the right atrium was cut open, and exsanguination was performed with intraventricular infusion of phosphatebuffered saline (PBS) until blood was completely flushed from circulation. This was followed by infusion of approximately 150 ml 4% paraformaldehyde (PFA) in PBS to fix the tissue. The brain was then removed from the skull and incubated overnight at 4°C in 4% PFA. The following day, brains were washed with PBS and incubated in 0.5 M sucrose for 24 hours. The 0.5 M sucrose solution was replaced with 1 M sucrose solution for another 24 hours at 4°C. Brains were then placed in Tissue Freezing Media (OCT Embedding Compound, Electron Microscopy Sciences, Hatfield, PA) and frozen by using liquid nitrogen and isopentane. Frozen samples were stored at −80°C. Brains were cut into 50-μm-thick sections by using a Leica cryostat at −20°C and collected in 0.1 M phosphate buffer (PB) with 0.1% sodium azide. All procedures were approved by the Harvard Animal Care and Use Committee.