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.
Johnson
I P
IP
Department of Anatomy and Developmental Biology, Royal Free and University College Medical School, Rowland Hill Street, NW3 2PF, London, UK. i.johnson@rfc.ucl.ac.uk
D000818:Animals; D001933:Brain Stem / Q000166:cytology / Q000473:pathology; D002452:Cell Count / Q000379:methods*; D005154:Facial Nerve / Q000166:cytology / Q000473:pathology; D020220:Facial Nerve Injuries / Q000473:pathology; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D018613:Microscopy, Confocal; D009046:Motor Neurons / Q000166:cytology / Q000473:pathology; D009474:Neurons / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012016:Reference Values; D013194:Staining and Labeling; D013997:Time Factors