At selected time points after induction of BRVO, eyes from euthanized mice were removed and fixed in 4% paraformaldehyde (pH 7.4) for 24 hours. Before removal from the animal, the orientation of the eyes was marked by placing a suture at the limbus and ensuring correct orientation of specimens during embedding, since BRVO was systematically induced in the superonasal quadrant. Eyes underwent routine paraffin processing, and tissue sections of 5-μm thickness were cut with a microtome (Leica Biosystems, Muttenz, Switzerland) and collected on slides. Tissue sections were deparaffinized and rehydrated before antigen retrieval with Tris–EDTA buffer (pH 9.0), as previously described in more detail elsewhere.
23 A rabbit polyclonal antibody against ionized calcium-binding adapter molecule 1 (anti-Iba1 rabbit polyclonal antibody, Wako Cat. No. 019-19741; Wako Pure Chemical Industries Ltd., Osaka, Japan) was applied overnight as the primary antibody to mark microglia. Visualization was achieved by incubation with a secondary goat anti-rabbit IgG (H+L), Alexa Fluor 594 conjugate (1:1000, A27016; Thermo Fisher Scientific. To stain Müller cells, an anticellular retinaldehyde-binding protein rabbit antibody (sc-28193; 1:300; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as primary antibody, followed by the secondary goat anti-rabbit antibody (see above). Slides were mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA), coverslipped, and examined under the microscope.
Immunohistochemical staining of protein adducts of pimonidazole in hypoxic cells was achieved using a 1:50 dilution of an anti-pimonidazole mouse IgG1 monoclonal antibody (hybridoma clone 4.3.11.3, Lot 9.7.11; Hypoxyprobe, Inc.) as the primary antibody overnight, followed by a goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 488 conjugate; 1:250, A11001; Thermo Fisher Scientific).
For preparation of retinal whole mounts, eyes were removed and fixed in 4% paraformaldehyde solution (pH 7.4) for 10 minutes. The anterior segment was removed, and the posterior portion, including the retina, was incubated for another 50 minutes in 4% paraformaldehyde (pH 7.4). Fixed retinas were mechanically detached from the choroid and extensively washed in 0.1% Triton in PBS followed by incubation in 5% normal goat serum (NGS) in 0.1% Triton in PBS for 2 hours at room temperature. Isolectin GS-IB4 from Griffonia simplicifolia (Alexa Fluor 647 conjugate; 1:100 in 5% NGS in 0.1% Triton in PBS; Thermo Fisher Scientific) and the rabbit polyclonal antibody against Iba-1 (see above) were used for double labeling of blood vessels and microglia cells, respectively. Retinas were incubated with primary antibodies for 48 hours at 4°C. A secondary goat anti-rabbit IgG (H+L) (Alexa Fluor 594 conjugate; 1:1000, A27016; Thermo Fisher Scientific) was used for the visualization of Iba-1 staining. In the chimera mice, a chicken polyclonal antibody against GFP (1:100, ab13970; Abcam, Cambridge, UK) was used for labeling of bone marrow–derived macrophages, followed by incubation with a preabsorbed goat polyclonal antibody to chicken IgY H+L (FITCH) (1:1000, ab7114; Abcam). Retinas were extensively washed in PBS–0.1% Triton, four radial cuts were made, and the retinas were flat mounted on a slide with the ganglion cell layer facing up. Whole mounts were coverslipped and examined under a confocal microscope.