Intracellular Localization Of CFH In The Mouse RPE

Abstract

Purpose: : Variant forms of complement factor H (CFH) play a central role in the pathogenesis of AMD. Previous studies have demonstrated that CFH is both synthesized and secreted from RPE cells. The intracellular localization of CFH, however, has not been shown although CFH has been reported to be secreted from both the apical and basolateral surfaces of the RPE. To refine the intracellular localization of CFH in vivo and document any age-related changes, we explored multiple methods of fixation and a variety of antibodies at various states of purification.

Methods: : BALB/c mice at various ages were purchased either from the NIA or The Jackson Laboratory. All research conducted was compliant with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Some mice were perfused with PBS containing 1 IU of heparin/ ml PBS before immersion fixation with 4% buffered paraformaldehyde. Other fixation protocols were examined including immersion fixation in Davidson’s fixative or snap freezing followed by acid methanol (97% methanol and 3% acetic acid) free substitution at -80^o C. All fixed specimens were embedded in paraffin. Immunohistochemistry was performed using a number of anti-CFH antibodies: Santa Cruz Biotechnology (catalog #sc-17951), Quidel antiserum (catalog #A312), and Novus Biologicals (catalog #NB100-62177). The whole antiserum from Quidel was additionally affinity purified using 100 ug human CFH (Sigma-Aldrich, catalog #C5813) linked to AminoLink Plus Coupling Resin (Pierce, catalog #20475).

Results: : Fixation with acid methanol at -80^o C or Davidson’s fixative yielded the best morphology including predominantly attached retina. Paraformaldehyde fixation yielded frequent retinal detachment. Immunolabeling of CFH with the Novus Biologicals or Quidel anti-CFH antibodies enabled the identification of a variety of intracellular vesicular structures within the RPE.

Conclusions: : Intracellular structures were best identified with fixation by snap freezing followed by a free substitution protocol with acetic acid/methanol. Affinity purification of individual antisera improved immunolabeling with selected antibody preparations. Intracellular vesicular structures in the cytoplasm of RPE cells appeared in aging mice.

Keywords: immunohistochemistry • retinal pigment epithelium • age-related macular degeneration