title: Localization of complement factor H gene expression and protein distribution in the mouse outer retina.
abstract: OBJECTIVE
  To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.
  
  
  METHODS
  Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.
  
  
  RESULTS
  Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.
  
  
  CONCLUSIONS
  Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.
authors: Department of Ophthalmology, University of California, Davis, CA.
authors: Zeljka
authors: Z
authors: Smit-McBride
authors: Department of Ophthalmology, University of California, Davis, CA.
authors: Sharon L
authors: SL
authors: Oltjen
authors: Jules Stein Eye Institute, University of California, Los Angeles, CA.
authors: Roxana A
authors: RA
authors: Radu
authors: Department of Human Anatomy and Cell Biology, University of California, Davis, CA.
authors: Jason
authors: J
authors: Estep
authors: Department of Ophthalmology, University of California, Davis, CA.
authors: Anthony T
authors: AT
authors: Nguyen
authors: Department of Human Anatomy and Cell Biology, University of California, Davis, CA.
authors: Qizhi
authors: Q
authors: Gong
authors: Department of Ophthalmology, University of California, Davis, CA.
authors: Leonard M
authors: LM
authors: Hjelmeland
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003180:Complement C3b Inactivator Proteins / Q000235:genetics* / Q000378:metabolism; D017242:Complement Factor H / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009928:Organ Specificity; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D003180:Complement C3b Inactivator Proteins; D012333:RNA, Messenger; D017242:Complement Factor H