May 2000
Volume 41, Issue 6
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Biochemistry and Molecular Biology  |   May 2000
Altered Expression of Secreted Frizzled-Related Protein-2 in Retinitis Pigmentosa Retinas
Author Affiliations
  • Stephen E. Jones
    From the British Retinitis Pigmentosa Society Laboratory, Department of Pharmacology, The Rayne Institute, GKT School of Biomedical Sciences, St Thomas’ Hospital, London, UK.
  • Catherine Jomary
    From the British Retinitis Pigmentosa Society Laboratory, Department of Pharmacology, The Rayne Institute, GKT School of Biomedical Sciences, St Thomas’ Hospital, London, UK.
  • John Grist
    From the British Retinitis Pigmentosa Society Laboratory, Department of Pharmacology, The Rayne Institute, GKT School of Biomedical Sciences, St Thomas’ Hospital, London, UK.
  • Hannah J. Stewart
    From the British Retinitis Pigmentosa Society Laboratory, Department of Pharmacology, The Rayne Institute, GKT School of Biomedical Sciences, St Thomas’ Hospital, London, UK.
  • Michael J. Neal
    From the British Retinitis Pigmentosa Society Laboratory, Department of Pharmacology, The Rayne Institute, GKT School of Biomedical Sciences, St Thomas’ Hospital, London, UK.
Investigative Ophthalmology & Visual Science May 2000, Vol.41, 1297-1301. doi:
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      Stephen E. Jones, Catherine Jomary, John Grist, Hannah J. Stewart, Michael J. Neal; Altered Expression of Secreted Frizzled-Related Protein-2 in Retinitis Pigmentosa Retinas. Invest. Ophthalmol. Vis. Sci. 2000;41(6):1297-1301.

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Abstract

purpose. Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive death of the photoreceptors due to apoptosis. To identify changes in gene expression associated with the degenerative state in RP retinas, expression profiling of apoptosis-related genes was performed using a gridded array technique.

methods. Total RNAs from RP and control retinas were used to generate radiolabeled cDNA probes to screen gridded membrane arrays of 205 apoptosis-related genes. Reverse transcription–polymerase chain reaction was used to generate probes corresponding to differentially expressed genes for Northern blot analysis and for mRNA in situ hybridization studies of retinal cryosections. Fluorescence immunocytochemistry was performed on retinal sections using available antibodies.

results. By expression profiling, we identified upregulated expression of the mRNA for secreted Frizzled-related protein-2 (SFRP2) in RP retina in comparison with control. By Northern blot analysis, SFRP2 mRNA levels were 2- to 20-fold higher in RP samples than in controls. The localization of SFRP2 mRNA by in situ hybridization varied according to the degree of degeneration, from stratified in relatively well-preserved retinas to diffuse in the highly degenerative state. By immunofluorescence, SFRP2 protein in RP retinas was found mainly to colocalize with the cell adhesion and signal transducing proteinβ -catenin.

conclusions. SFRPs can regulate apoptosis in vitro and appear to interact with the Wnt/Frizzled signaling pathway, which includes routes to apoptotic activation. Increased SFRP2 expression in RP retinas suggests that an altered pattern of Wnt signal transduction may be a step in the degenerative process linking causal mutations with eventual photoreceptor demise.

Accumulating evidence indicates a common mechanism responsible for photoreceptor cell death in many forms of inherited retinal degeneration, including those classed as retinitis pigmentosa (RP). 1 2 We obtained first indirect evidence that photoreceptor death in RP is apoptotic in nature, through detection of increased mRNA levels of the marker gene clusterin in affected retinas. 3 Subsequently, several groups directly demonstrated activation of apoptosis in photoreceptors during retinal degeneration in different rodent models of RP. 4 5 6 Apoptosis is a term covering a multitude of cellular pathways, which, after activation, converge upon an apparently rather uniform collapse of cell integrity. Although transgenic studies have attempted to determine the roles of specific genes in rodent photoreceptor apoptosis, 7 8 9 10 as yet no clear picture has emerged to indicate which potential pathways of apoptotic activation occur in different instances of photoreceptor cell death. 
To gain insight into mechanisms of human retinal degeneration, we undertook expression profiling to identify further differentially expressed apoptotic genes in RP, using screenings of gridded gene arrays. We report patterns of retinal expression of one such gene, SFRP2, and discuss some implications in relation to possible apoptotic mechanisms. 
Methods
Tissues
Control eyes from donors with no history of ocular disease were obtained from the UK Transplant Support Service, Bristol, Moorfields Eye Hospital, London, or St. Thomas’ Hospital, London. Eyes from RP donors were obtained through the British RP Society’s Eye Donor Scheme. In all cases, written informed consent was obtained before enucleation. The postmortem interval (pmi) between death and freezing of dissected retinas in liquid nitrogen or fixation of whole eyes in 4% paraformaldehyde was recorded where possible. Sectors of fixed tissue were embedded, frozen in liquid nitrogen–cooled isopentane and stored in liquid nitrogen until use. Sections (10-μm-thick) were cut on a cryostat, thaw-mounted on gelatin-coated slides, and stored at− 70°C until used. 
For control eyes, the donor details were as follows: CON1, male, age 53 years, pmi ∼34 hours; CON2, female, 76 years, pmi = 11 hours; CON3, female, 57 years, pmi = 29 hours; CON4, female, 72 years, pmi = 28 hours; and CON5, male, 79 years, pmi = 21.5 hours. For RP eyes, the donor details were as follows: RP1, male, 60 years, pmi = 7.5 hours, advanced sporadic RP; RP2, male, 84 years, pmi = 15 hours, probable recessive RP (six brothers in family, of whom two were affected); RP3, male, 51 years, pmi ∼10 hours, probable X-linked RP (two brothers, of whom one was affected); and RP4, male, 79 years, pmi not greater than 24 hours, simplex RP. 
RNA Extraction and Northern Blot Analysis
Total RNA was extracted from frozen tissues using the RNeasy system (Qiagen GmbH, Hilden, Germany) or according to a protocol associated with the differential array screening procedure (Clontech Laboratories, Palo Alto, CA). For Northern blot analysis, 3 μg RNA samples were processed as previously described. 3 Isolated cDNA inserts or polymerase chain reaction (PCR) products (∼20–60 ng) in low melting point agarose (GIBCO–BRL Life Technologies, Paisley, UK) were labeled with α-[32P]dCTP using the Rediprime kit (Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer’s instructions, and hybridizations were performed in the presence of 50% formamide at 42°C overnight, followed by stringent washing and autoradiography. The probes were stripped off between hybridizations. Quantification of autoradiographic signals was performed using a laser densitometer (LKB, Sweden). 
Differential Screening of Gridded Gene Arrays
Gridded arrays of 205 apoptosis-related partial cDNAs were obtained from Clontech Laboratories and processed in accordance with the manufacturer’s protocols. Total RNA samples (2 μg) from CON1 and RP3 retinas were reverse-transcribed in the presence ofα -[32P]dATP to produce complex probes. After hybridization to the membranes at 68°C overnight, stringent washings, and autoradiography, the patterns of spots were compared by direct visual inspection of the films and after computer-based image enhancement. Genes were then identified by reference to a key and to the online database at<http://www.clontech.com/archive/JUL98UPD/JUL98Atlas.html>. 
Generation of cDNA Clones and Probes
Approximately 1 μg of retinal total RNA (RP3) was reverse-transcribed in 20 μl reactions with 1.25 U AMV reverse transcriptase (GIBCO–BRL Life Technologies) and random hexamer primers (Pharmacia, St. Albans, UK) for 60 to 90 minutes at 42°C, followed by heat inactivation of the enzyme. PCR amplification of an SFRP2 partial cDNA was then performed using primers HSARP1.1, 5′-GGGTCGCGCCCACGATGCTG-3′, and HSARP1.2, 5′-CTTCCTCGGTGGCTGGCAGG-3′ (derived from the human sequence, GenBank Accession Number AF017986), in 50 μl reactions containing 10 pmol of each primer, 200 mM dNTPs, and 1.0 U Taq DNA polymerase (GIBCO–BRL Life Technologies), with the following conditions: 94°C for 3 minutes, then 30 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 60 seconds, and a final step of 72°C for 5 minutes. A PCR product of the expected size (510 bp) was cloned into the pCR2.1-TOPO vector (Invitrogen, Leek, The Netherlands) according to the manufacturer’s protocol to give clone pHSARP1.TA27. For the generation of riboprobes, the SFRP2 insert was subcloned into the vector pBluescript SK (Stratagene, La Jolla, CA) to give clone pBS.SARP1. Cloning and orientation of the expected SFRP2 cDNAs were confirmed by partial sequence analysis (MWG-Biotech GmbH, Ebersberg, Germany). The control probe was a PCR product of 835 bp amplified from human retinal cDNA using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPD): GAPDH.1, 5′-CCTTCATTGACCTCAACTACAT-3′, and GAPDH.2, 5′-TACCAGGAAATGAGCTTGACAA-3′, based on the published sequence, 11 GenBank Accession No. M33197. 
In Situ Hybridization
Labeling of sense and antisense riboprobes generated from linearized restriction endonuclease enzyme digestions of the pBluescript clone of SFRP2, and subsequent hybridization of the probes to cryostat sections of human retinas, was performed using the color in situ kit (Amersham Pharmacia Biotech) in accordance with the manufacturer’s recommendations. After hybridization overnight at 55°C in the presence of 50% formamide, stringent washing, and RNaseA treatment, the hybridized probes were detected by incubation with alkaline phosphatase–conjugated anti-fluorescein antibody and development in the presence of NBT/BCIP substrates. A deep purple-brown reaction product indicates hybridization. 
Immunocytochemistry
Cryostat sections (10 μm) of fixed frozen tissues were processed using the classic immunofluorescence technique. 12 For detection of SFRP2, the primary antiserum was a goat polyclonal anti–FRP-2 (sc-7426; Santa Cruz Biotechnology, Santa Cruz, CA), used at a dilution of 1:300. The secondary antibody, a fluorescein isothiocyanate–coupled anti-goat IgG (Sigma, St. Louis, MO), was used at 1:200. For detection of β-catenin, the primary antiserum was a goat polyclonal antibody (sc-1496; Santa Cruz Biotechnology) used at 1:300, with the secondary used as above. 
Results
Differential Screening of Gridded Gene Arrays
Hybridization of single-stranded retinal cDNA probes from donors CON1 and RP3 to gridded arrays of human apoptosis-related genes produced differing autoradiographic patterns (data not shown). Three genes showing increased expression in RP were identified as those encoding c-Jun, PIG7 (manuscript in preparation) and secreted apoptosis-related protein-1 (SARP1), 13 also known as secreted Frizzled-related protein-2 (sFRP-2), 14 and SDF5. 15 We have adopted the approved nomenclature SFRP2 for this gene 16 and report further analysis of its expression in normal and RP retinas. 
Northern Blot Analysis of SFRP2 Expression in the Retina
We examined the retinal expression of SFRP2 mRNA in 4 cases of RP and 3 controls (Fig. 1 , top panel). A major transcript of approximately 2.4 kb was detectable in all RP samples, together with a likely precursor mRNA of ∼4.4 kb in the most highly expressing cases; by visual inspection, no corresponding bands were detectable in control samples. For the cases of RP, in which the retinas exhibited different stages of histomorphologic degeneration (see below), SFRP2 mRNA levels were variably increased compared with controls. However, there were no obvious correlations between the SFRP2 mRNA level and the extent of degeneration or RP type. Probing for GAPD expression confirmed that the differences were not due to variation in RNA loading or integrity (Fig. 1 , middle panel). Laser densitometric analysis permitted detection of weak signals corresponding to the SFRP2 mRNAs at ∼2.4 kb in control samples. After normalization to GAPD levels, comparisons indicated a range of approximately 2- to 20-fold increased expression of SFRP2 mRNA in RP retinas relative to that in controls (Fig. 1 , bottom panel). 
In Situ Hybridization Detection of SFRP2 mRNA
We investigated the cellular distribution of SFRP2 mRNA expression in available retinal sections using a color in situ hybridization protocol (Fig. 2) . Positive hybridization was indicated by the presence of a purple reaction product; careful examination permitted discrimination of this product from pigment in the retina or retinal pigment epithelium (RPE). In a normal retina (CON5), mRNA was detectable at the outer margins of the inner nuclear layer (INL), with a slightly stronger reactivity at the inner segments of the photoreceptors (PR; Fig. 2A ). Similar patterns were detected in sections from two additional control retinas (data not shown). There was some evidence of nonspecific reactivity with the sense probe in CON5 (Fig. 2B) . In sections of retina from RP1 (Fig. 2C) , which showed severe disruption of retinal architecture, loss of photoreceptors, gliosis, and extensive accumulation of pigment within the retina, there was evidence of diffuse labeling throughout the retina, including in association with pigment clumps. Although this case showed the highest level of expression of SFRP2 mRNA by Northern blot analysis (Fig. 1 , top, lane 1), the intensity of in situ hybridization labeling in the sections examined was not as great as with case RP3 (Fig. 2E) , suggesting regional variability in SFRP2 mRNA levels within the retina. In the macular region of retina RP3 (Fig. 2E) , which showed moderate preservation of retinal morphology, including the presence of photoreceptor rosettes, a more intense labeling was associated with the INL and also the residual photoreceptor nuclei, but most strikingly very intense staining was detected at the ganglion cell layer (GCL; arrowheads). In retina RP4 (Fig. 2G) , which showed extensive regions of relatively well preserved retinal morphology, including surviving photoreceptors, there was labeling in the INL and outer nuclear layer (ONL) and also at the photoreceptor inner segments, resembling that seen in normal retinas. The sense control probings of RP retinas (Figs. 2D 2F 2H) showed only slight background coloration. 
Immunocytochemical Analyses
SFRP2.
The pattern of immunofluorescence using a polyclonal anti-SFRP2 antibody was compared in sections of control and RP retinas (Figs. 3A 3C 3E 3G 3I ). Very little immunostaining was evident with the control retina (Fig. 3A ; near the inner retinal margins there is nonspecific fluorescence from the vasculature) or in sections from two additional controls (data not shown); occasional amacrine cells were faintly positive. In the three cases of RP examined, there were differences in the distribution of immunofluorescence. In retinas RP1 and RP3 (Figs. 3C and 3E , respectively), most of the labeling was located at the retinal margin of the inner limiting membrane (ILM). In retina RP4 (Fig. 3G) , however, more intense labeling was noted in specific retinal layers, notably the GCL, the inner plexiform layer, the outer plexiform layer and amacrine cells, the photoreceptor outer segments, and also, most probably, the RPE. Interestingly, distinct cytoplasmic labeling was noted in numerous residual surviving photoreceptors of the ONL. 
β-Catenin.
In view of the implicated role of SFRPs in regulating the expression ofβ -catenin via the Wnt signaling pathway 12 13 (see also Discussion), we examined the pattern of β-catenin immunoreactivity on adjacent retinal sections (Figs. 3B 3D 3F 3H 3J) . In control retina, the outer limiting membrane, outer and inner plexiform layers, and (at a lower intensity) the GCL were immunopositive (Fig. 3B) ; similar results were obtained with two additional controls (data not shown). In retinas RP1 and RP3, the immunolabeling was localized mainly to the ILM (Figs. 3D 3F) , although with evidence of labeling also at Bruch’s membrane seen most clearly in RP3 (Fig. 3F , arrow), and diffuse labeling throughout the body of the degenerative retina. There was some indication that this labeling was cytoplasmic in the ONL, INL, and GCL of RP3 (Fig. 3F) . In retina RP4, the pattern of localization (Fig. 3H) was similar to that observed with normal retina, although with a greater intensity and with additional labeling of the ILM. 
Control sections lacking primary antibodies (Figs. 3I 3J) displayed minimal nonspecific immunofluorescence, mainly associated with the retinal vasculature and choroid. The RPE and some intraretinal pigment granules showed a yellow autofluorescence. 
Discussion
By examination of apoptosis-related genes in human retinal degeneration using differential screening of gridded arrays, we identified increased expression of the mRNA for SFRP2, a member of the family of secreted Frizzled-related proteins, in RP retinas. The SFRP genes, of which human, rodent, bovine, and Xenopus members have been described, 16 encode secreted proteins with cysteine-rich domains homologous to those of Frizzled (Fz) proteins but lacking the membrane-spanning segments of the latter group. The numerous Frizzled genes encode cellular membrane proteins with seven transmembrane domains, which act as receptors for the similarly diverse array of secreted Wnt proteins. 16 17 18 Through signaling interactions conserved between Drosophila and vertebrates, Wnts have been implicated in the control of key developmental processes via regulation of cell morphogenesis, cell mitogenesis, and cell fate. SFRPs may modulate signaling via Fz through their binding interactions with Wnts, or indeed via direct binding to Fz receptors themselves. 19 A recent report described SFRP2 mRNA localization to the INL of bovine retina and SFRP5 expression in the RPE, suggesting possible roles for these genes in regulating the polarity of photoreceptors or other retinal cells. 20 Our present findings add to the evidence for a role for SFRP2 in the eye, potentially in influencing retinal cell survival. 
In the cases of RP examined, we found increased and altered patterns of expression of SFRP2 mRNA and protein relative to controls, although there was variability in both the levels and distribution of transcripts and immunoreactive protein. SFRP2 protein localization correlated most closely with the mRNA pattern of expression in the case of RP4, where the retina was relatively well preserved. In the other RP cases compared, with more advanced degeneration, the majority of immunoreactive protein was concentrated at the inner margins of the ILM, possibly in association with the terminal end feet of Müller glial cells which proliferate during degeneration, suggesting that as the disease progresses SFRP2 is increasingly vectorially secreted out of the neural retina into the vitreous. The discrepancy between high SFRP2 mRNA levels and relatively low levels of immunoreactive SFRP2 protein, notably in retinas RP1 and RP3, may further support this possibility, although more information on the relationship between transcription, translation, and posttranslational processing and degradation of SFRP2 will be required to establish this. 
The role of SFRPs in modulating or antagonizing the Wnt signaling pathway, taken together with our present findings, suggests that dysregulation of this pathway may be occurring in the apoptotic degenerative processes in RP retinas. Of potential relevance are reports of increased expression of SFRP homologues during apoptosis occurring in vitro 13 21 and in involution of rat mammary gland, ovary, and prostate. 22 Furthermore, overexpression of transfected SFRP2 in breast adenocarcinoma cells increases their resistance to apoptotic signals, associated with increased intracellular levels of β-catenin, 13 a multifunctional protein engaged in cell–cell junction interactions with E-cadherin and transcriptional regulatory complexes with members of the TCF/Lef-1/pangolin family. 17 Immunoreactive SFRP2 andβ -catenin largely colocalized in RP retinas, but we found no clear evidence of increased β-catenin levels, suggesting that if modulation of Wnt signaling is occurring in retinal degeneration, the interplay of regulatory interactions is more complex than in in vitro situations. Speculatively, SFRP2 upregulation in RP retinas may reflect an anti-apoptotic response, analogous to that proposed for the glycoprotein clusterin, which ultimately fails to protect the photoreceptors due to loss of regulation of the Wnt signaling pathway and downstream collapse of apoptosis suppression mechanisms. Although the complex network of Wnt signaling and its ramifications over a diversity of cellular interactions ensures that progress in unraveling the significance of altered SFRP expression in RP will not be straightforward, potentially new therapeutic targets may emerge for the eventual treatment of retinal degenerative diseases. 
 
Figure 1.
 
Results from Northern blot analysis of SFRP2 mRNA expression in RP and control retinas. Total RNA samples (∼3 μg): Lanes 1, RP1; 2, RP2; 3, RP3; 4, RP4; 5, CON2; 6, CON3; and 7, CON4. Top panel, probed with SFRP2 cDNA; middle panel, same blot reprobed with GAPD cDNA. Approximate transcript sizes are shown in kilobases. Bottom panel shows ratio of mRNA levels as determined by laser densitometry (peak values of the 2.4 kb transcripts).
Figure 1.
 
Results from Northern blot analysis of SFRP2 mRNA expression in RP and control retinas. Total RNA samples (∼3 μg): Lanes 1, RP1; 2, RP2; 3, RP3; 4, RP4; 5, CON2; 6, CON3; and 7, CON4. Top panel, probed with SFRP2 cDNA; middle panel, same blot reprobed with GAPD cDNA. Approximate transcript sizes are shown in kilobases. Bottom panel shows ratio of mRNA levels as determined by laser densitometry (peak values of the 2.4 kb transcripts).
Figure 2.
 
In situ hybridization analysis of SFRP2 mRNA expression in RP and control retinal cryosections using antisense (A, C, E, G) and sense (B, D, F, H) probes. Retinal sections from donors CON5 (A, B); RP1 (C, D); RP3 macular region (E, F); RP4 (G, H). Arrowheads (E) indicate strong labeling of ganglion cell layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium. Scale bar, 100 μm.
Figure 2.
 
In situ hybridization analysis of SFRP2 mRNA expression in RP and control retinal cryosections using antisense (A, C, E, G) and sense (B, D, F, H) probes. Retinal sections from donors CON5 (A, B); RP1 (C, D); RP3 macular region (E, F); RP4 (G, H). Arrowheads (E) indicate strong labeling of ganglion cell layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium. Scale bar, 100 μm.
Figure 3.
 
Fluorescence immunocytochemical detection of SFRP2 (A, C, E, G, I) and β-catenin (B, D, F, H, J) expression in control and RP retinas. Retinal sections from donors CON5 (A, B), RP1 (C, D), RP3 (E, F), and RP4 (G, H). Sections from CON5 but lacking primary antibody indicate nonspecific fluorescence of the RPE and choroid (I, J). Arrow (F) indicates Bruch’s membrane. Scale bar, 100 μm.
Figure 3.
 
Fluorescence immunocytochemical detection of SFRP2 (A, C, E, G, I) and β-catenin (B, D, F, H, J) expression in control and RP retinas. Retinal sections from donors CON5 (A, B), RP1 (C, D), RP3 (E, F), and RP4 (G, H). Sections from CON5 but lacking primary antibody indicate nonspecific fluorescence of the RPE and choroid (I, J). Arrow (F) indicates Bruch’s membrane. Scale bar, 100 μm.
The authors thank the Rayne Management Committee for the provision of research facilities. 
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Figure 1.
 
Results from Northern blot analysis of SFRP2 mRNA expression in RP and control retinas. Total RNA samples (∼3 μg): Lanes 1, RP1; 2, RP2; 3, RP3; 4, RP4; 5, CON2; 6, CON3; and 7, CON4. Top panel, probed with SFRP2 cDNA; middle panel, same blot reprobed with GAPD cDNA. Approximate transcript sizes are shown in kilobases. Bottom panel shows ratio of mRNA levels as determined by laser densitometry (peak values of the 2.4 kb transcripts).
Figure 1.
 
Results from Northern blot analysis of SFRP2 mRNA expression in RP and control retinas. Total RNA samples (∼3 μg): Lanes 1, RP1; 2, RP2; 3, RP3; 4, RP4; 5, CON2; 6, CON3; and 7, CON4. Top panel, probed with SFRP2 cDNA; middle panel, same blot reprobed with GAPD cDNA. Approximate transcript sizes are shown in kilobases. Bottom panel shows ratio of mRNA levels as determined by laser densitometry (peak values of the 2.4 kb transcripts).
Figure 2.
 
In situ hybridization analysis of SFRP2 mRNA expression in RP and control retinal cryosections using antisense (A, C, E, G) and sense (B, D, F, H) probes. Retinal sections from donors CON5 (A, B); RP1 (C, D); RP3 macular region (E, F); RP4 (G, H). Arrowheads (E) indicate strong labeling of ganglion cell layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium. Scale bar, 100 μm.
Figure 2.
 
In situ hybridization analysis of SFRP2 mRNA expression in RP and control retinal cryosections using antisense (A, C, E, G) and sense (B, D, F, H) probes. Retinal sections from donors CON5 (A, B); RP1 (C, D); RP3 macular region (E, F); RP4 (G, H). Arrowheads (E) indicate strong labeling of ganglion cell layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium. Scale bar, 100 μm.
Figure 3.
 
Fluorescence immunocytochemical detection of SFRP2 (A, C, E, G, I) and β-catenin (B, D, F, H, J) expression in control and RP retinas. Retinal sections from donors CON5 (A, B), RP1 (C, D), RP3 (E, F), and RP4 (G, H). Sections from CON5 but lacking primary antibody indicate nonspecific fluorescence of the RPE and choroid (I, J). Arrow (F) indicates Bruch’s membrane. Scale bar, 100 μm.
Figure 3.
 
Fluorescence immunocytochemical detection of SFRP2 (A, C, E, G, I) and β-catenin (B, D, F, H, J) expression in control and RP retinas. Retinal sections from donors CON5 (A, B), RP1 (C, D), RP3 (E, F), and RP4 (G, H). Sections from CON5 but lacking primary antibody indicate nonspecific fluorescence of the RPE and choroid (I, J). Arrow (F) indicates Bruch’s membrane. Scale bar, 100 μm.
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