Fixed tissue was cryoprotected and serially sectioned at a thickness of 10–20 μm. The tissue sections were immunolabeled using 6E10 antibody (Covance), anti-calbindin D28k (Sigma-Aldrich), anti-GluR1 (from Millipore or developed as described [88 (link)]), anti-LAMP1 (BD Pharmingen; San Jose, California, USA), anti-cathepsin B (Millipore), anti-synaptophysin (Millipore), and antibodies that selectively label Aβ38 and Aβ42 (Covance). Immunofluorescence analyses used appropriate Invitrogen secondary antibodies (Thermo Fisher Scientific; Waltham, Massachusetts, USA), and images were captured with a Zeiss fluorescence microscope system (Carl Zeiss, Inc.; Thornwood, New York, USA) and with a Nikon C2 point-scanning confocal microscope with NIS-Elements AR software (Nikon Instruments; Melville, New York, USA). Other images were produced via avidin–biotin–peroxidase protocols (Vector Laboratories; Burlingame, California, USA) that used 3,3′-diaminobenzidine as the chromogen. In each case, treatment groups were immunostained together and analyzed under the same instrument settings. Equally spaced coronal sections along the rostral–caudal axis of the hippocampus were used to determine the average immunoreactivity intensity and area of staining across four different view-fields.
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