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Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

Smit-McBride Z, Oltjen SL, Radu RA, Estep J, Nguyen AT, Gong Q, Hjelmeland LM - Mol. Vis. (2015)

Bottom Line: Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of California, Davis, CA.

ABSTRACT

Purpose: 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.

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In situ hybridization in the C57BL/6, BALB/c, 129/Sv, and 129/Sv Cfh−/− mouse eye. A C57BL/6 eye sectioned near the optic nerve with its negative control (A), RPE65 positive control (B), and Cfh probe (C) are shown. Similar in situ hybridization (ISH) results were observed in the BALB/c strain: negative control (D), RPE65 positive control (E), and Cfh probe (F). Note the robust Cfh mRNA signal in the RPE in both strains (Figure 4C,F). Negative and RPE65 positive controls and Cfh ISH results for a 129/Sv Cfh−/−mouse (H, J, L) and its background strain, 129/Sv (G, I, K) are shown. To visualize the Fast Red signal masked by the RPE pigment, the Fast Red fluorescence property was used to image the sections using a rhodamine filter for 129/Sv (M) and 129/SvCfh −/− (N). Note the greatly reduced signal in the RPE of the Cfh−/− eye compared to the background strain. RPE=retinal pigment epithelium. Scale bar=20 μm.
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f4: In situ hybridization in the C57BL/6, BALB/c, 129/Sv, and 129/Sv Cfh−/− mouse eye. A C57BL/6 eye sectioned near the optic nerve with its negative control (A), RPE65 positive control (B), and Cfh probe (C) are shown. Similar in situ hybridization (ISH) results were observed in the BALB/c strain: negative control (D), RPE65 positive control (E), and Cfh probe (F). Note the robust Cfh mRNA signal in the RPE in both strains (Figure 4C,F). Negative and RPE65 positive controls and Cfh ISH results for a 129/Sv Cfh−/−mouse (H, J, L) and its background strain, 129/Sv (G, I, K) are shown. To visualize the Fast Red signal masked by the RPE pigment, the Fast Red fluorescence property was used to image the sections using a rhodamine filter for 129/Sv (M) and 129/SvCfh −/− (N). Note the greatly reduced signal in the RPE of the Cfh−/− eye compared to the background strain. RPE=retinal pigment epithelium. Scale bar=20 μm.

Mentions: To define the cell types in the posterior pole expressing Cfh transcripts, we performed in situ hybridization. The ISH results using the RNAscope 2.0 FFPE Assay are shown in Figure 4. In the RPE of the C57BL/6 and BALB/c mouse, the negative control probe DapB showed no signal (Figure 4A,D). The positive control, a probe for RPE65, showed a robust signal in the RPE as expected (Figure 4B,E). The Cfh message was expressed mainly in the RPE with negligible expression in the choroid (Figure 4C,F). In addition, no message was detected in the photoreceptors (data not shown).


Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

Smit-McBride Z, Oltjen SL, Radu RA, Estep J, Nguyen AT, Gong Q, Hjelmeland LM - Mol. Vis. (2015)

In situ hybridization in the C57BL/6, BALB/c, 129/Sv, and 129/Sv Cfh−/− mouse eye. A C57BL/6 eye sectioned near the optic nerve with its negative control (A), RPE65 positive control (B), and Cfh probe (C) are shown. Similar in situ hybridization (ISH) results were observed in the BALB/c strain: negative control (D), RPE65 positive control (E), and Cfh probe (F). Note the robust Cfh mRNA signal in the RPE in both strains (Figure 4C,F). Negative and RPE65 positive controls and Cfh ISH results for a 129/Sv Cfh−/−mouse (H, J, L) and its background strain, 129/Sv (G, I, K) are shown. To visualize the Fast Red signal masked by the RPE pigment, the Fast Red fluorescence property was used to image the sections using a rhodamine filter for 129/Sv (M) and 129/SvCfh −/− (N). Note the greatly reduced signal in the RPE of the Cfh−/− eye compared to the background strain. RPE=retinal pigment epithelium. Scale bar=20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4323684&req=5

f4: In situ hybridization in the C57BL/6, BALB/c, 129/Sv, and 129/Sv Cfh−/− mouse eye. A C57BL/6 eye sectioned near the optic nerve with its negative control (A), RPE65 positive control (B), and Cfh probe (C) are shown. Similar in situ hybridization (ISH) results were observed in the BALB/c strain: negative control (D), RPE65 positive control (E), and Cfh probe (F). Note the robust Cfh mRNA signal in the RPE in both strains (Figure 4C,F). Negative and RPE65 positive controls and Cfh ISH results for a 129/Sv Cfh−/−mouse (H, J, L) and its background strain, 129/Sv (G, I, K) are shown. To visualize the Fast Red signal masked by the RPE pigment, the Fast Red fluorescence property was used to image the sections using a rhodamine filter for 129/Sv (M) and 129/SvCfh −/− (N). Note the greatly reduced signal in the RPE of the Cfh−/− eye compared to the background strain. RPE=retinal pigment epithelium. Scale bar=20 μm.
Mentions: To define the cell types in the posterior pole expressing Cfh transcripts, we performed in situ hybridization. The ISH results using the RNAscope 2.0 FFPE Assay are shown in Figure 4. In the RPE of the C57BL/6 and BALB/c mouse, the negative control probe DapB showed no signal (Figure 4A,D). The positive control, a probe for RPE65, showed a robust signal in the RPE as expected (Figure 4B,E). The Cfh message was expressed mainly in the RPE with negligible expression in the choroid (Figure 4C,F). In addition, no message was detected in the photoreceptors (data not shown).

Bottom Line: Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of California, Davis, CA.

ABSTRACT

Purpose: 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.

Show MeSH
Related in: MedlinePlus