Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: Immunofluorescence labeling of PFA-fixed wild type and C3 -/- mouse eyecup tissue with A) anti-C3 (MP Biomedicals, 55463) and B) anti-C3d (R&D Systems, AF2655) antibodies, showing genuine antibody labeling in the RPE of wild type samples, localized primarily to the basal side of the RPE. Note suspected non- specific binding of the anti-C3 primary antibody to photoreceptor outer segments (OS). Abca4 -/- samples were included as a no primary control, to show general tissue AF. Labeling using the anti-C3 antibody was performed using frozen sections and labeling using the anti-C3d antibody was performed using paraffin-embedded tissue sections. General tissue AF at 488 nm was included to assist in identifying eyecup structures and for comparison to immunofluorescence signal in the no primary control conditions. Note that each C3 antibody may detect multiple C3 fragments. The scale bar represents 10 µm and is consistent across all images. Choroid, RPE and OS are shown. Arrowheads show red blood cells, which are fluorescent at 488 nm in paraffin sections. Arrows show the location of suspected RPE basal labyrinth.

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Immunofluorescence, Labeling, Antibody Labeling, Binding Assay, Control, Comparison

Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: A) Representative images showing immunofluorescence labelling in PFA-fixed wild type, Abca4 -/- , Cfd -/- and Abca4 -/- ; Cfd -/- mouse eyecup tissue sections, using anti-C3 (MP Biomedicals) and anti-C3d (R&D Systems) antibodies. Anti-C3 immunofluorescence was performed using 6-12-mo frozen sections and anti-C3d immunofluorescence was performed using 12-mo paraffin embedded sections. For each image, 488 nm AF (green) and C3 immunofluorescence (magenta) are shown together, to aid in localizing the immunofluorescence signal. C3 immunofluorescence is also shown on its own in greyscale. Choroid, RPE and photoreceptor outer segments (OS) are labelled, with dotted lines indicating the basal (top) and apical (bottom) boundaries of the RPE. Arrowheads show the location of Bruch’s membrane, between the RPE basal labyrinth and the choroid region. Scale bar represents 10 µm. B-C) Quantification of total RPE immunofluorescence signal in each genotype using the anti-C3 and anti-C3d antibodies, respectively. Total immunofluorescence was quantified by measuring the mean immunofluorescence signal in the RPE of 3-4 images minus the fluorescent signal in 3-4 no primary antibody control images, per mouse. Each data point represents one mouse. Mean +/- 95% C.I. are shown. Data were analyzed by 1-way ANOVA with Tukey’s multiple comparisons test. **** indicates p<=0.0001

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Immunofluorescence, Membrane, Control

Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: A) Schematic representation of production and breakdown of the C3 protein. Estimated fragment molecular weights under reducing conditions are shown. Western blot was performed using eyecup lysate (RPE, choroid, sclera) detected using an antibody made against the C3d region (anti-C3d). Yellow shows the C3d region of the protein, which is part of the C3 α-chain (C3α). Note that the C3d region of the C3 protein contains the thioester site, which participates in covalent binding to target molecules (yellow arrowheads), potentially affecting fragment molecular weight of the C3b, iC3b, C3dg and C3d α-chain. B) Qualitative western blot images showing labelling of wild type and C3 -/- eyecup tissue using the anti-C3d antibody. Marked regions i-vi indicate C3 fragments of interest and are shown zoomed-in in greyscale in panel (C) with predicted C3 fragment identities indicated.

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Western Blot, Binding Assay, Molecular Weight

Journal: bioRxiv

Article Title: Loss of Complement Factor D suppresses alternative pathway activation but fails to reduce lipofuscin accumulation in the retinal pigmented epithelium of Abca4 -/- mice

doi: 10.1101/2025.10.25.684461

Figure Lengend Snippet: A) Representative blots labelled with the anti-C3d antibody, showing all genotypes. Note the faint C3 α-chain fragment detected with the anti-C3d antibody, which may be the opsonized iC3b α62 fragment (ops iC3b) and was frequently detected in Cfd +/+ but not Cfd -/- conditions. Arrowhead (*) indicates the suspected opsonized C3b α-chain fragment selected for quantification. Higher molecular weight opsonized C3bα fragments were not quantified due to transfer inconsistencies and blot damage, which frequently affected the high molecular weight region of the blots. Rpe65 and β-actin were included as loading controls. B) Quantification of various C3 fragments under reducing conditions from mouse eyecup lysate, detected by the anti-C3d antibody. Animals used in this experiment range from 6-12 months of age. Relative quantities of C3 preprotein, C3α, iC3(H2O) α72, opsonized C3bα (*), iC3b α62 and C3dg were normalized to Rpe65 and β-actin, which were shown to have stable signal across the experimental conditions of interest (Supplementary Figure 6). Given the large number of experimental conditions, samples were run across multiple blots with a mix of genotypes per blot. To control for variability between blots, normalized C3 fragment quantities were expressed relative to the C3α fragment from WT samples. Note that quantities of C3α and iC3(H2O) α72 (filled circles) are represented on the left y-axis, and quantities of the preprotein, opsonized C3bα, iC3b α62 and C3dg fragments (empty circles) are represented on the right y-axis. Data were analyzed using a two-way ANOVA, revealing an effect of C3 fragment (p<0.0001), genotype (p<0.0001) and C3 fragment X genotype (p<0.0001). Multiple comparisons were performed using Tukey’s test, and p-values <0.05 are shown on the graph. C-D) Analysis of data from D, organized by independent variable (Abca4 genotype and Cfd genotype, respectively). Within each C3 fragment, data are represented relative to the respective control conditions ( Abca4 +/+ or Cfd +/+ ). Data were analyzed using a two-way ANOVA, revealing an effect of C3 fragment X genotype for the Cfd genotype (p<0.0001) but not Abca4 genotype (p=0.6). Multiple comparisons were performed using Šídák’s test, and p-values <0.05 are shown on the graph. Each data point is a measurement from a single animal, and data are represented as mean +/- 95% C.I. for all graphs.

Article Snippet: C3 labeling was quantified in mouse eyecup structures, such as the RPE and choroid, using antibodies generated against full-length mouse C3 protein (referred to as “anti-C3”, MP Biomedicals) or purified mouse C3d (referred to as “anti-C3d”, R&D Systems) ( ).

Techniques: Molecular Weight, High Molecular Weight, Control