Computational protocol: Inhibition of the classical pathway of the complement cascade prevents early dendritic and synaptic degeneration in glaucoma

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Protocol publication

[…] The protocol for immunohistochemical staining of retinal sections was identical for both mice and rats except where stated otherwise. Mice were sacrificed by cervical dislocation, their eyes enucleated and placed in 4 % PFA ON. For rats, a rising concentration of CO2 was used, with cervical dislocation confirming death. Following this, eyes were cryoprotected in 30 % sucrose, frozen in OCT and cryosectioned at 12 μm. Eyes from the following number of animals (and central retina sections) were used: (in mice [animals (sections)]; 4mo D2 n = 6[], 9mo D2 n = 3[], 4mo D2-Gpnmb+n = 9[], 9mo D2-Gpnmb+n = 5[], 4mo D2.C1qa n = 5[], 9mo D2.C1qa n = 3[]. Rats: normotensive n = 9[], normotensive plus C1 inhibitor n = 3[], OHT n = 3[], OHT plus C1 inhibitor n = 3[]). Central retina sections were brought to RT for 1 h, permeabilized with 0.1 % Triton-X for 30mins, blocked with 5 % chick serum in PBS and stained ON at RT in primary antibody (for mice; rabbit anti-PSD-95; 51-6900, Life Technologies, USA, or goat anti-IBA1; ab5076, Abcam, UK; for rats; rabbit anti-PSD-95; ab18258, Abcam, UK). After primary antibody incubation, sections were washed 5 times in PBS, stained for 4 h at RT with secondary antibody (for mice; goat anti-rabbit AF488; Life Technologies, USA, for rats; goat anti-rabbit AF555, Abcam, UK). Slides were then washed a further 5 times with PBS, stained with DAPI (mice) or Hoechst 33342 (rats) for 15mins, mounted with fluoromount, coverslipped and sealed with nail-polish. For PSD95 quantification in DBA/2 J mice and rats, images of central retina showing the IPL and ONL were collected under identical conditions on a Leica SP8 confocal microscope and Leica DM6000B respectively (image area = 553.57 μm2). Fluorescent intensity of antibody staining was quantified using ImageJ. Three-dimensional reconstruction of IBA1+ cells was performed using IMARIS. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Rattus norvegicus
Diseases Glaucoma, Retinal Degeneration, Retinal Diseases, Retinitis, Neurodegenerative Diseases