The eyes were collected at different time points following RD and fixed in 2% paraformaldehyde (Sigma-Aldrich, Dorset, UK) for 2 h at room temperature post-enucleation. The eyes were then washed with PBS and placed in 10%, 20% and 30% sucrose (Sigma-Aldrich, USA) before embedment in OCT (optimal cutting temperature) compound and cryo-sectioned using Leica CM 1900 cryostat (Leica Microsystems, Milton Keynes, UK) at 16 μm thickness. H & E and immunofluorescent staining was performed following published protocols [18 (link), 19 (link)] and antibodies used were listed in Table 1 . A negative control, without the primary antibody, was carried out in each staining.
https://imagej.net/Fiji/ ). Briefly, the number of CD68+ immune cells, DAPI-labelled nuclei and cone-arrestin+ photoreceptor cells were counted using a multi-point tool in Fiji. The number of rod cells was calculated by subtracting the number of cone-arrestin+ cells from the total DAPI+ cells in the ONL [20 (link)]. Synaptophysin+ areas in the OPL and IPL areas were measured using a free-hand selection tool in Fiji. GFAP quantification was achieved by counting GFAP-positive fibres within the INL layer and post the INL layer of the retina. The data were presented as mean ± standard error of the mean (SEM), normalised to 100 μm of retinal length.
Antibodies used for immunohistochemistry and western blot
| Antibody | Dilution | Company | Catalogue number |
|---|---|---|---|
| CD68 | 1:300 | BioRad | MCA1957 |
| Cone-arrestin | 1:1000 | Millipore | AB15282 |
| GFAP | 1:200 | DAKO | Z0334 |
| GS | 1:2000 | Sigma | G2781 |
| IL-33 | 1:50 | R & D | AF3626 |
| Donkey anti-goat IgG Alexa Fluor 488 | 1:300 | Jackson Immunoresearch | 705–545-147 |
| Donkey anti-rabbit IgG Alexa Fluor 488 | 1:300 | Jackson Immunoresearch | 711–585-152 |
| Goat anti-rat IgG Alexa Fluor 594 | 1:300 | Invitrogen | A-11007 |
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