Our study was performed on three phenotypically healthy subjects: two of them were Caucasian with Alzheimer familiarity (48 and 55 years old) and the other one was Asian without Alzheimer familiarity (45 years old) and affected by an adenoviral keratoconjunctivitis at the moment of the withdrawal. Patients were examinated with fundus camera to highlight retinal plaques as described in literature; only the Caucasian patient presented a strong alteration in the posterior chamber with numerous plaques on retina (fig. a 1 and 2). In this present work, human tear samples were examinated with ICC using anti-Amyloid Beta X-42 antibody; samples were stained with DAB if positive, no staining if negative. (Fig. B 1,2 and 3). NaCl solution was employed as negative control and protein Amyloid Beta-42 as positive control (Fig. C 1 and 2). Our test was not influenced by irritative and infective phenomena that can occur in tissue during the execution of withdrawal.
Tear’s sample were dispensed on Thermo Scientific adhesion slides, dried at 76°C for 4 hours and hydrated with 100% ethanol for 3 minutes, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes and distilled water for 3 minutes. The area of interest was marked using a PAP pen, which draws an hydrophobic barrier to prevent the waste of reagents by keeping liquid pooled in a small droplet. The endogenous peroxidases were neutralized using peroxidase block (3–4% v/v hydrogen peroxide) for 10 minutes, followed by protein block for 10 minutes (0.4% casein in phosphate-buffered saline, with stabilizers, surfactant and 0.2% bronidox L as preservative) to reduce non-specific binding of primary antibody and polymer. Samples were incubated with the primary antibody (anti-Amyloid Beta X-42, clone 12F4, a purified mouse monoclonal IgG1k in buffer containing 0.1 M Tris-Glycine pH 7.4, 150 mM NaCl with 0.05% sodium azide, Millipore) diluted 1:200 for 60 minutes. Post primary (rabbit anti-mouse IgG < 10µg/mL in 10% v/v animal serum in tris-buffered saline/0.09% Proclin 950) was incubated for 20 minutes, followed by Novolink Polymer (anti-rabbit Poly-HRP-IgG < 25µg/mL containing 10% v/v animal serum in tris-buffered saline/0.09% Proclin 950) for 20 minutes. To avoid the presence of residual reagent from the previous step, starting from peroxidase block, each step was interspersed with a washing with wash buffer (diluted 1:10, < 1%-2-Methyl-2H-Isothiazol-3-One) for 5 minutes. Peroxidase activity was developed with DAB working solution (DAB chromogen 1.74% v/v 3,3’-diaminobenzidine, in a stabilizer solution) diluted 1:20 in DAB substrate buffer and after 5 minutes the excess of reagent was washed with distilled water for 5 minutes. Samples were counterstained with hematoxylin for 30 seconds, washed again with running water for 5 minutes and distilled water for 3 minutes, and dehydrated with 70% ethanol for 3 minutes, 95% ethanol for 3 minutes and 100% ethanol for 3 minutes. Finally, they were covers lipped with synthetic mounting medium and results were interpreted using a light microscope.
Immunocytochemistry (ICC) allows the identification by light microscopy of an antigen and its location in cells through specific antigen-antibody reaction. In our study, we employed ICC indirect method: the specific antigen was recognized by an unlabeled primary antibody which binds the secondary antibody (or post primary), conjugated to the horseradish peroxidase (HRP or polymer) which reacts with the substrate yielding a chromogenic development at the antigen site. Samples were counterstained with hematoxylin, covers lipped and results were interpreted at light microscopy.