Neuropathological diagnosis of AD was made according to NIA-AA criteria. anti-APP antibodies yielded signal in ganglion cells, amacrine cells, horizontal cells and Mller cells in both control and AD cases. We observed small extracellular deposits positive for anti-A antibodies 12F4 and 6E10 and negative for 4G8 and curcumin. A subset of Bay 59-3074 these deposits could be characterized as corpora amylacea. In conclusion we found that retinal manifestations of AD pathology appear to be different compared to cerebral AD pathology. Using a qualitative cross-sectional approach, we did not find A/APP related differences in the retina between AD and control subjects. In contrast, tau related changes were found to be present in cases with cerebral AD pathology, suggesting retinal tau as a potential biomarker for AD. Alzheimers disease, Healthy control Tissue processing Within 12?h post-mortem, eyes were removed. The anterior parts of the eye, including the cornea and lens, were dissected and the eyecup was filled with tissue-tek (cat# 4583, Sakura). Eyes were snap frozen using iso-pentane at ??100?C and stored at ??80?C. Eyes were defrosted in 4% PFA at room temperature for 48?h prior to dissection. The eye was dissected in four quadrants through the vertical and horizontal meridian resulting in naso-superior, naso-inferior, temporal-superior and temporal-inferior quadrants containing retinal tissue from macula to ora serrata (Fig.?1). Quadrants were dehydrated prior to embedding in paraffin according to the following protocol: 3?h formalin 4% at 35?C, 1?h ethanol 70% 35?C, 1?h ethanol 80% 35?C, 1?h ethanol 96% 35?C, 3 times 1?h alcohol 100% 35?C, 3 times 1?h xylene 35?C, 4 times 1?h paraffin 62?C. Paraffin embedded tissue was sectioned using a microtome at 5-m and 10-m thickness and mounted on TOMO slides (cat# TOM-1190, Matsunami). Mounted slides were dried overnight at 37?C prior to staining. Per patient, at least 25 sections per region were stained with APP/A antibodies to overcome sampling bias. Open in a separate window Fig. 1 Processing of post-mortem eyes. Anterior parts of eyes were removed (a). Formalin fixed eyes were dissected through the horizontal (b) and vertical meridian (c). Superior (red, arrows) and nasal (green, arrows) parts were cut in CD48 10?m sections from anterior to posterior. As a result, sections contained all retinal layers from ora serrata to the posterior pole: retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), Bay 59-3074 inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photoreceptors (PR), retinal pigment epithelium (RPE), choroid and sclera Immunohistochemistry?(IHC) Immunohistochemistry was initially performed on 5 and 10?m thick eye sections. As both thicknesses yielded comparable results, 10?m sections were therefore used for the full cohort. Sections were deparaffinized and endogenous peroxidase activity was suppressed with 0.3% H2O2 in phosphate buffered saline (PBS) for 30?min. Antigen retrieval was performed with 10?mM/L pH?6.0 sodium citrate buffer heated by autoclave. Sections were incubated overnight at room temperature with primary antibody, diluted in antibody diluent (cat# kpxxabb500, immunologic). For primary antibodies details and dilutions, see Table?2. Omission of the primary antibodies was taken along as negative controls. Positive controls consisted of 5?m thick paraffin sections of hippocampal regions of AD patients. Sections were incubated for 30?min with envision (cat# 5007, DAKO). 3,3-Diaminobenzine (DAB) was used for color development. Nuclear counterstaining consisted of Mayers hematoxylin. Sections were dehydrated and cover slipped using quick-D (cat# 7281, Klinipath). Table 2 Primary antibodies used in Bay 59-3074 this study Alzheimers disease, Healthy control In summary, diffuse phosphorylated tau for three phosphorylation sites was observed in AD, with a predilection for the peripheral retina, while NFTs, neuritic plaques, fibrillar tau or paired helical filaments were not detected. Discussion In this post-mortem study of well-characterized AD and control cases, we qualitatively assessed antibody panels for APP, A and tau on AD and control retinal cross-sections. We found that diffuse phosphorylated tau in the retina separated AD cases from controls.