Immunohistochemistry
Formalin-fixed paraffin-embedded hippocampal tissue blocks were sectioned at 5 mm thickness and subjected to fluorescence immunohistochemistry. Tyramide signal amplification (TSA) was used for double fluorescence immunostaining of the same tissue section (Tóth & Mezey, 2007). Double staining was used in combination with antibodies against neuron-specific nuclear protein (NeuN; Cell Signaling Technology, #24307) and Aβ peptide 1-42 (Abcam, ab201061), and a combination of antibodies against microtubule association protein-2 (MAP2, MILLIPORE, #1990899) and p-Tau (phosphor T231, Abcam, ab151559). All samples were processed using standard protocols and solutions (Tóth & Mezey, 2007).
Aβ and p-Tau depositions were quantified using an image analysis system (AON-STUDIO, 2021) based on the contrast between the stained and unstained regions. In each case, images were captured, and a threshold optical density was obtained that differentiated positively stained tissue from the background. Image analysis was performed while blinded to the identity of each patient. Three regions (×200) of the hippocampus were randomly sampled from each patient. The final measurement was obtained by dividing the area of the immune-positive plaques by the total sampling area. Data from all sampled areas were used for group comparisons. The significance of group differences was determined using the Mann-Whitney U test.