Cryostat

For histological analysis, eyes (n = 20) from mice that did not receive eye drops after blast wave exposure were post-fixed, bisected, embedded in resin, sectioned on a microtome, and stained with toluidine blue (Fisher, Waltham, Massachusetts, United States). Representative images were collected on an Olympus Provis AX70 (Olympus, Center Valley, Pennsylvania, United States) with a 60x oil objective lens. To quantify retinal pigment epithelium (RPE) damage, a grading scale was developed to classify the vacuoles: 1 (normal, very infrequent and small), 2 (small and infrequent), 3 (small and frequent), 4 (large and infrequent), and 5 (large and frequent). The number of pyknotic nuclei in the outer nuclear layer (ONL) or inner nuclear layer (INL) was quantified within a single section of retina through the middle of each eye.
For immunohistochemistry, eyes (n = 65) were cryoprotected in 30% sucrose overnight at 4°C, embedded in Tissue Freezing Medium (Triangle Biomedical, Durham, North Carolina, United States) and then sectioned on a cryostat (Fisher, Pittsburgh, Pennsylvania, United States). Sections of 10-μm thickness were collected in round on 12 slides, such that each slide contained representative sections from the entire eye.

For immunohistochemistry, Bl/6 (n = 32) and D2 (n = 33) eyes and olfactory epithelium (Bl/6 n = 4) were cryo-protected in 30% sucrose overnight at 4°C, embedded in Tissue Freezing Medium (Triangle Biomedical, Durham, NC) and then sectioned on a cryostat (Fisher, Pittsburgh, PA). We collected 10 μm-thick sections on 12 slides, with each slide containing representative sections from the entire eye. The sections were rinsed with PBS and incubated at room temperature in normal donkey serum at 1:20 in 0.1 M phosphate buffer with 0.5% bovine serum albumin and 0.1% Triton X 100 (PBT) for 2 hours, followed by an overnight incubation at 4°C in primary antibody in PBT (Table 1). The sections were rinsed with PBS and incubated with a secondary antibody at a 1:200 dilution (Life Technologies, Grand Island, NY) for 2 hours at room temperature, rinsed with PBS and mounted in Vectashield Mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Imaging was performed on a Nikon Eclipse epifluorescence microscope (Nikon, Melville, NY) or an Olympus FV-1000 confocal microscope (Olympus, Center Valley, PA). Imaging on the Olympus FV-1000 microscope was performed through use of the VUMC Cell Imaging Shared Resource. The tissue was assessed in a masked fashion.

Embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) overnight and submerged in 12.5%, 25% sucrose, and 25% sucrose/OCT solutions sequentially. Fixed embryos were embedded in OCT compound (Tissue-Tek) and sectioned at 12 μm thickness using a cryostat (Fisher Scientific). Tissue slides were kept at -20°C. In situ hybridization was performed with digoxygenin-labeled Myh9, Myh10, Myh14, and Myl9 antisense or sense probes according to standard methods. Immunostaining was performed using the following primary antibodies: anti-rabbit NMHCIIA (Covance, 1:500), Phalloidin-conjugated Alexa 488 (1:200), anti-rat E-cadherin (Invitrogen, 1:300), anti-rabbit p63 (Abcam, 1:200), anti-rabbit Ki67 (Thermo Scientific, 1:200), and anti-rabbit cleaved caspase-3 (Asp175) (Cell signaling, 1:200). Anti-rabbit IgG-Cy3, anti-rat Cy2, or Dylight 649 (Jackson Immunoresearch Lab) were used as secondary antibodies to visualize the signals. Images were captured using a Zeiss Axio Imager Z2 microscope or a Zeiss Cell Observer spinning disk confocal microscope.

Six weeks after immunization, optic nerves and eyes were enucleated and fixed in 4% paraformaldehyde for 2 h (optic nerves) or 1 h (eyes). Thereafter, the tissues underwent a 30% sucrose treatment and got embedded in a Neg-50 compound (Tissue-Tek; Fisher Scientific). Longitudinal sections of the optic nerve (4 µm) and cross-sections of the retina (10 µm) were cut with a cryostat (Fisher Scientific) for further staining (57 (link)).