Retina Whole Mount Staining and Measurement of Neovascularization

Retinal whole mount staining was performed as previously described.^15,16 The eyes of normal mice were enucleated at P7 and P10 and were fixed in 4% paraformaldehyde for 30 minutes at room temperature. We removed the corneas and lenses first, then the eyecups were fixed in 4% paraformaldehyde for 10 more minutes. The retinas were carefully dissected and washed three times in phosphate-buffered saline solution (PBS) for 15 minutes. Retinal flat mounts were stained at 4°C overnight in 100 µL PBS containing 1% bovine serum albumin (BSA) and 1% Triton X-100 with at least one of the following primary antibodies: Lectin-FITC (L9381, 1:50, Sigma-Aldrich Corp., St. Louis, MO, USA) or Collagen IV (BP8019,1:100, Acris, Herford, Germany). Afterward, the following secondary antibodies, goat anti-rabbit 594, (1:200, Invitrogen, Carlsbad, CA, USA) were added for two hours in the same buffer. Then, the flat mounts were mounted on microscopic slides and cut into four quadrants. Finally, microscopic images (original magnification × 16) were taken of the stained samples using a fluorescent microscope (Leica DMRBE, Bensheim, Germany) which was equipped with the IM50 software. The vessel area of P7 and P10 were measured with image processing software Cell F (Olympus, Hamburg, Germany).

To analyze the development of superficial retinal vessels at P7 and “both superficial and deep retinal vessels” at P10, images were taken with the Olympus BX51 microscope. Six to eight fields (original magnification × 200) between the arterioles and venules were randomly chosen. The growth of vessel network was evaluated by measuring capillary density (vessel/retinal area ratio) and width, which were quantified using Cell F software (Olympus).

Numbers of tip cell were counted manually by counting the numbers of sprouts at P7 (original magnification × 200) or branchpoints at P10 (original magnification × 100) in six to eight fields for each retina under the fluorescence microscope. The length of tip cell was measured from the edge of the bottom area to the end of the sprout at P7 (original magnification × 400) and at P10 (original magnification × 200), which were quantified using Cell F software (Olympus).

We defined the zones imitating the classification of ROP. “Central zone” is defined as a circle, centered on the disc, whose radius is 500 µm from the disc; “Middle zone” is from the edge of “Central zone” to the circle, whose radius is 1000 µm from the disc; “Peripheral zone” is the remaining area out of circle of 1000 µm from the disc (Fig. 1).

The schematic representation of the central, middle and peripheral retinal zones in mice. “Central zone” is defined as a circle, centered on the disc, whose radius is 500 µm from the disc; “Middle zone” is from the edge of “Central zone” to the circle, whose radius is 1000 µm from the disc; “Peripheral zone” is the remaining area out of circle of 1000 µm from the disc. C, central zone; M, middle zone; P, peripheral zone.