Fig 1: Microglia and phospho-tau detected in brain tissue collected from the cortex of a 51-year-old female Asian zoo elephant (Elephas maximus). Tissue stained using immunohistochemistry and counterstained with cresyl violet, (A) IBA1 (brown, 1:1,000, Fujifilm, 019–19,741), (B–D) CP13 (brown, 1:1,000, Gift from Dr. P. Davies). Images (A–D) were taken on an Axiophot brightfield microscope (Carl Zeiss Microscopy, Jena, Germany), with a 10x/0.32 Plan-Apochromat objective. (E–F) Tissue was stained with CP13 (red) and GFAP (green, 1:1,000, Abcam., ab68428) using immunofluorescence (note that blood vessels appear in green due to autofluorescence). Images (E–F) were taken on a CLSM 780 confocal microscope (Carl Zeiss Microscopy, Jena, Germany), using a 20x/0.8 DICII objective and DPSS 561–10 diode and Argon lasers at excitation wavelengths of 555 and 488 nm. Confocal stacks in layers II and III of the cerebral cortex were imaged at 512 × 512 pixel resolution with a z-step of 1 µm for a pinhole setting of 1 Airy unit. Images are presented as maximum intensity projections of the Z-stack, made using ZenBlue (version 3.3, Carl Zeiss Microscopy, Jena, Germany). All scale bars are 50 µm. In (A), microglia are evenly distributed and ramified without any activated (ameboid) forms. In (B, C), neurons stained with tau were found in layers III and II, respectively. In (D) tau “speckling” is visible in brown throughout layer III, and it is also visible in (E–F) in red. Images (E–F) show tau-positive neurons (thick arrowheads), and fibrils in red, and astrocytes in green (as well as autofluorescent blood vessels which are much thicker). Astrocytic end feet are visible around some blood vessels (thin arrows). In (F), astrocytes are indicated with stars, note their presence around the tau-positive neuron.