Five-by-three tiled confocal images over the cross-section of the third ventricle were acquired on a Zeiss LSM880 microscope at an image depth of 8 bit. Confocal micrographs were loaded in Fiji 1.52e, and their signal intensity for either pERK1/2 or VEGFA was quantified in pre-defined tanycyte subgroups in male mice kept at either 25 °C or 40 °C. Images were acquired at identical settings (including laser power output, digital gain/offset) to allow for comparisons be made on signal intensities between the experimental groups.
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