Imager a2 microscope

Specimens were examined histologically for evidence of intimal formation utilizing 5-μm hematoxylin and eosin (H&E)-stained cross sections. Five to six equally-spaced sections from each of the 4 regions were stained from each animal. Digital images of stained sections were collected with light microscopy using a Zeiss Imager-A2 microscope (Hallbergmoos, Germany). At each location the luminal area, intimal area, medial area, and circumference were determined using ImageJ software (NIH; Bethesda, MD). In addition, when applicable, the intima-to-media area ratio (I/M), percent of the arterial wall composed of the intima (intimal area/intimal area + medial area or I/I+M), and percent stenosis (intima area/intimal area + lumen area * 100) were calculated.

Brains (n=3/time point) were embedded into a single gelatin block (Multiblock_ Technology; Neuroscience Associates). Cryosections containing all study brains were mounted and immunostained for ionized calcium binding adaptor molecule 1 (Iba-1), to identify all microglia. The immunostained slides were imaged using a Zeiss Imager A2 microscope with AxioCam MRc5 digital camera. Images were rotated 90 degrees to the left. These sections were obtained from the same brains processed for aminocupric silver staining above. Data from these Iba-1 stained sections have been published for this and other brain regions^{52 (link),75 (link),77 (link),78 (link)}.

Tissue sections were prepared as described above for histology and then antigen retrieved by boiling for 10 min in sodium citrate (10 mM, pH 6.0) (Vector Laboratories). The tissues were incubated in 1% H2O2 for 30 min to quench the endogenous hydroperoxide activities. M.O.M kit (Vector Laboratories) was used for immunostaining for NFATC1 antibody (BD Pharmingen, 556,602). The brown color was developed using DAB kit (Vector Laboratories). The tissues were then blocked with 5% normal horse serum in PBS before being incubated with PECAM1 antibodies (Santa Cruz Biotechnology, sc-1506) and secondary anti-bodies. The red color was developed using Vector Red Kit (Vector Laboratories). Immunofluorescence staining of NOTCH1 was performed on frozen sections. The embryos were collected at E10.5 and fixed in 4%PFA at 4 °C for one hour, soak in 15% and 30% sucrose sequentially and embedded in OCT compounds with orientation for front sections at 8 μm on the positive charged slides. Tissue sections were then post-fixed in the cold ethanol and acetone (1:1) solution for 5 min and stored at − 80 °C. Tissue sections were air dried for 45 min before staining. The tissues were then blocked with 5% normal horse serum in PBS before being incubated with NOTCH1 antibodies (R&D, AF5267) and secondary antibodies. Stained tissue sections were photographed using a Zeiss Imager A2 microscope.