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.