“…However, there are also limitations of the IF: the use of antibodies against nuclear proteins (to distinguish neurons from non‐neuronal cells) does not identify cell types among the non‐neuronal cells, NeuN antigens are not expressed by a small number of neuronal populations (Mullen et al, ), and only regions and volumes of tissues that can be dissected macroscopically can be analyzed (Lent et al, ). Automated versions of the IF have been reported, both for the homogenization procedure (Azevedo et al, ) and for the counting procedure, using flow cytometry (Collins et al, ; Young et al, ; Herculano‐Houzel et al, ). Long‐standing concerns about loss of nuclei when using a biochemical homogenization approach (Brizzee et al, ; Hadjiolov et al, ; Lovtrup‐Rein and McEwen, ; Cragg, ; Kato and Kurokawa, ; Clarke and Oppenheim, ; Yuhas and Jabr, ; Carlo and Stevens, ; Verkhratsky and Butt, ; Charvet et al, ) have recently been addressed and dispelled in two studies that directly compared the IF, in side‐by‐side experiments, with results obtained by stereology (Bahney and von Bartheld, ; Miller et al, ).…”