Quantification of NeuN immunopositive neurons in the white matter. (A) A surgically-resected temporal lobe from a patient with epilepsy who had undergone standard temporal lobectomy. (B) A 7μm-thick section was cut, immunolabelled using the neuronal marker, NeuN, and scanned using the whole slide scanner (LEICA). Using WSA, the white matter was selected automatically or manually on digitised images of NeuN immunopositive sections. In WSA automated, the programme identified the white matter by following a series of algorithms such as tissue and background separation (C), and showed the training step, whereby the user selected ‘segments’ that were representative of the cortex (orange) or white matter (yellow; D), and the programme learned to identify other ‘patches’ of similar features (E). (F) In WSA manual, the user interactively outlined the white matter. After ROI selection, NeuN immunopositive cells in the white matter (G) of all cases were quantified by WSA (H). (I) In SA, images (yellow) were manually acquired by the user and then fed into a semi-quantitative analysis programme for quantification. (J) For stereology, a small area in the deep white matter was selected by the user and the programme moved through each point within the selection to allow the user to count NeuN immunopositive cells at 63× magnification. Scale bar = 1 cm (A), 2 mm (B-F, I-J), 200 μm (G-H).