Rapid estimates of neuron number in the confocal microscope combined with in situ hybridisation and immunocytochemistry

A rapid counting protocol is described which combines the optical disector and Cavalieri methods on non-embedded slices of fixed tissue viewed in the confocal laser scanning microscope. By eliminating the embedding stage and avoiding the need to align adjacent sections in the z plane for counting, considerable time savings are gained over physical disector methods. It also allows the remaining non-embedded sections to be used for other purposes, such as in situ hybridisation and immunocytochemistry. Starting with fixed brainstem, it was possible in less than 2 h to determine the total number of motoneurons in both facial nuclei of an adult Sprague-Dawley rat. This method revealed that the normal facial nucleus contained approximately 3200 motoneurons (n = 12 rats). One month following facial nerve avulsion (n = 4 rats), mean numbers of motoneurons were reduced by 75%. Using intervening sections, changes in neuronal number were compared with changes in in situ hybridisation signal and immunostaining.