Ultravision mouse tissue detection system anti mouse hrp dab

Following sacrifice, brain tissue was dissected and fixed in 10% formalin overnight before paraffin embedding. Immunohistochemical staining was completed at the UMMS Morphology Core using anti-ionized calcium-binding adapter molecule (IBA1) antibody (Wako; 1:1000) and subsequently labeled with streptavidin-biotin immunoenzymatic antigen for detection with 3,3′-diaminobenzidine (DAB) (UltraVision Mouse Tissue Detection System Anti-Mouse HRP/DAB; Lab Vision). Images were acquired from the described CNS areas by light microscopy (cortex; CA1, CA3, and DG of the hippocampus) at × 40 magnification for process length and cell body size measurements of microglia using ImageJ. Cell process length for each microglial cell was measured by tracing all extensions off of the soma to their distal termination using ImageJ’s freehand measuring tool. For each microglia, the length of all processes was summed to obtain the total cell process length. The soma area was measured by tracing the perimeter of the cell body and measuring the contained area using ImageJ’s freehand tracer and the area measurement function. Microglia were analyzed from five to nine images taken randomly from each CNS region from each mouse. The investigator was blinded to the sample groups during staining, image acquisition, and ImageJ analysis. IBA1 positivity was measured using the Color Deconvolution plug-in in ImageJ.

OilRed O tissue staining on OCT-embedded frozen liver sections was completed and quantification was performed using ImageJ to assess hepatic steatosis. Formalin-fixed paraffin-embedded liver sections were stained with anti-mouse myeloperoxidase antibody (Abcam) and subsequently labeled with streptavidin-biotin immunoenzymatic antigen for detection with 3,3’-diaminobenzidine (DAB) (UltraVision Mouse Tissue Detection System Anti-Mouse HRP/DAB; Lab Vision).