Abstract
Purpose: :
Our objectives were to identify the immunolocalization of complement factor H (Cfh) in the mouse retina and RPE/choroid as well as the levels of expression of Cfh and Cfhr2 message in the aging mouse eye. Because in vitro studies have demonstrated apical secretion of CFH from the RPE, we were specifically interested in the apical region of the RPE in vivo.
Methods: :
BALB/c mice were purchased from The Jackson Laboratory (2 months old) and the NIA (18 months old). All research was compliant with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were perfused with PBS containing 1 IU of heparin/ ml PBS under deep anesthesia. Eyes were then enucleated and snap frozen, followed by freeze substitution in acid methanol (97% methanol and 3% acetic acid). All fixed specimens were embedded in paraffin. Immunolabeling was performed using either goat α-mouse Factor H or rabbit α-mouse Factor H (Santa Cruz Biotechnology). EDTA mouse plasma (Innovative Research) was enriched for low abundant proteins using a Seppro Mouse Spin Column (Sigma-Aldrich) and then used for Western blot analysis. Purified human CFH (Complement Technology, Inc.) and mouse Cfh (gift from Claire Harris, Cardiff University) were used as positive controls. Cfh and Cfhr2 gene expression in young and old male and female RPE/choroid and retina was evaluated by qPCR .
Results: :
Both antibodies consistently labeled photoreceptors for Cfh protein, but RPE cytoplasmic staining was inconsistent. Western Blots: Both antibodies recognized purified human and mouse CFH, as well as Cfh in mouse plasma. qPCR: Cfh and Cfhr2 (Cfhrb) were detected in both the RPE/choroid and retina of young and old male and female mice. Cfhr1, Chfr3 and Gm4788 (Cfhrc) were not detected in preliminary work and so were not included in this study. The most significant change observed was an upregulation of Cfhr2 in the retina of older female mice.
Conclusions: :
Immunolabeling: Both antibodies detected Cfh in photoreceptors, but only the rabbit Ab detected Cfh in RPE cytoplasm of some eyes. Western Blots: rabbit α-mouse Factor H Ab gave a stronger detection signal. qPCR: Even though we tested all the Cfh-related genes expressed in the mouse liver, we were only able to detect significant expression of Cfh and Cfhr2 (Cfhrb) in both the RPE/choroid and retina. Only one significant age related change was observed, an upregulation of Cfhr2 in the retina of old female mice.
Keywords: immunohistochemistry • gene/expression • retinal pigment epithelium