Fig 2: Multi-label immunofluorescence reveals tau positive interfascicular glia are mature oligodendrocytes, not oligodendrocyte precursor cells, astrocytes or microglia, in adult rat brains. (A,B) To determine whether interfascicular and perineuronal glial cells containing tau are mature oligodendrocytes, tissue sections were dephosphorylated and stained with multi-label immunofluorescence for DAPI (nuclear counterstain, blue), Tau1 (mid-tau antibody, green), myelin basic protein (MBP, mature oligodendrocyte marker, red), and oligodendrocyte lineage transcription factor 2 (Olig2, an oligodendrocyte marker, cyan). In the corpus callosum (A) and hippocampal pyramidal cell layers (B), the Tau1-positive glial cells were also stained with MBP and Olig2 indicating they are mature oligodendrocytes. MBP labeling in tau/Olig2-positive cells was evident, but not all tau/Olig2-positive cells were clearly labeled with MBP suggesting either levels of cytoplasmic MBP were below detection or a subset of unidentified Olig2-positive cells express tau. (C,D) To further explore if the tau-positive glial cells were oligodendrocyte precursor cells, tissue sections were stained with multi-label immunofluorescence for DAPI (blue), Tau1 (green), neural/glial antigen 2 (NG2, an oligodendrocyte precursor marker, red), and Olig2 (cyan). In the corpus callosum (C) and hippocampal pyramidal cell layers (D), the Olig2-positive cells that were NG2-positive showed little to no tau reactivity indicating that the tau-positive glial cells are not oligodendrocyte precursor cells. Some oligodendrocytes were Olig2-positive but negative for tau and NG2. (E,F) To establish whether tau-positive glial cells were astrocytes or microglia, tissue sections were stained with multi-label immunofluorescence for DAPI (blue), Tau1 (green), ionized calcium-binding adapter molecule 1 (IbaI, a microglial marker, red) and glial fibrillary acidic protein (GFAP, an astrocyte marker, cyan). In the corpus callosum (E) and hippocampal pyramidal cell layers (F), the tau-positive cells showed little to no co-labeling with GFAP-positive cells or IbaI-positive cells indicating that the tau-positive glial cells are not astrocytes or microglia. Representative cells of each phenotype are indicated with yellow arrows in images. Scale bars in merged images are 10 µm in panels (A,C,E) and 20 µm in panels (B,D,F).