Integrated optical density (IOD) analysis

Computer-assisted image analysis software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD, USA) was used to analyze the IOD of menin-positive immunostaining detected by light microscopy (Olympus BX-URA2, Olympus Corporation, Tokyo, Japan) and double-positive immunostaining of menin/NeuN detected by fluorescence microscopy (Olympus IX81). IOD=Density(mean) * Area. The same software was also used to quantify total neurons and menin-positive neurons detected by fluorescence microscopy (Olympus IX81). We selected nine SIV-infected macaques as the infected group and compared them with four uninfected macaques (control group) for further statistical analysis of IOD. Five microscopic images (original magnification: 400×) were acquired from layers II to V of the frontal cortex from each of the thirteen selected macaques. The menin and NeuN double-labeled expression levels were scaled with a positive-stained IOD of each image using Image Pro Plus software, version 6.0 (Media Cybernetics). We also chose five images with double-NeuN/menin staining from each macaque to quantify total NeuN-positive and menin-positive neurons, as well as the mean values. We also semi-quantitatively evaluated the IHC results using the Image-Pro Plus program (Media Cybernetics). Menin-positive cells were quantified in 10 random 200× light microscopic fields of cortical layers 2–5 in the middle frontal gyrus. These findings showed an increased number of cells expressing menin, when more than 700 menin-positive cells were counted. Background levels were obtained in tissue sections immunostained in the absence of a primary antibody; corrected optical density = optical density − background. We also performed semiquantitative assessments for the following IHC findings: NeuN expression, astrocytic gliosis, caspase 3-positive cells.