Fig. 4

Images in (A) show representative images of human hippocampal sections from three AD cases and three NC (Group 1 A, 1B) immunohistochemically stained against IRIS-1 and IRIS-2. The graphs in (B, C) demonstrate significantly lowered IRIS-1 of IRIS-2 immunoreactivity in the CA1 region of AD compared to NC (each dot represents the mean value of 6 images with 10–12 neurons used for quantification from every image. Each group consists of n = 3 cases). In (D) representative images of the CA1 region in three T2D cases and three NC are shown (Group 2). The graph in (E, F) shows the significantly lowered IRIS-1 of IRIS-2 immunoreactivity in T2D cases compared to NC (each dot represents the mean value of 6 images with 10–12 neurons used for quantification from every image. Each group consists of n = 3 cases). Statistical analysis of changes between NC vs. AD and NC vs. T2D was performed using two-tailed student T-tests, with error bars indicating standard deviation (SD). Of note, the difference in immunoreactivity seen in (A and D) is due to the different postfixation methods used after autopsy, where tissue from cases in Group 1 A and B (A) were immersion fixed in PFA directly after autopsy, while tissue from cases in Group 2 (C) was first snap frozen, followed by immersion fixation in PFA. Scale bars = 50 μm. Images in (G, I) show representative immunofluorescence stainings against IRIS-1 and IRIS-2 (red), tau phosphorylated at Ser 202/ Thr205 (AT8) (green) and DAPI (blue) of sections from the AD cases included in Group B (n = 7 AD cases). IRIS-1 (G) and IRIS-2 (I) positive neurons in the CA1 regions was divided into low, intermediate or high AT8 immunoreactivity, where low represents healthy neurons (very low or no AT8 staining), and high represents neurons with high amount of AT8 immunoreactivity (each representative neuron is indicated with dotted rectangles, green for low, yellow for intermediate and red for high, n ≥ 30 neurons per case and n ≥ 10 neurons per AT8 immunoreactivity category was analyzed). The thickness of the Z-stack varies between 19.8 and 39.6 μm. Scale bars = 10 μm. Dotplots for all neurons analysed were included and color coded by case number, with the estimate of the fixed effect as the slope, shown in (H) for IRIS-1 and (J) for IRIS-2