A simple method for coupling in situ hybridization and immunocytochemistry: application to the study of peptidergic neurons.
We have devised a simple procedure for immunostaining of sections that have previously undergone autoradiographic visualization of mRNAs by in situ hybridization. Classical hybridocytochemistry techniques were performed first on cryostat sections of formaldehyde-fixed tissue. Standard methods were used for slide coating by emulsion dipping and for revelation, fixation, and coverslipping steps. The key to this method is the emulsion removal, or permeabilization, by a short trypsin incubation (0.2% for 20-30 sec) which facilitates the good access of antibodies used in a subsequent immunocytochemical technique to section epitopes. Usual immunofluorescence and immunoperoxidase procedures were successfully performed after this treatment. The immunoreactivity of several neuropeptides was well preserved after this procedure. In addition to its usefulness in our studies, this general method should be applicable to many other situations in which autoradiographic and immunocytochemical detections must be coupled.
Bugnon
C
C
Laboratoire d'Histologie-Embryologie-Cytogénétique, CNRS URA 561, Université de Franche-Comté, Faculté de Médecine, Besançon, France.
Bahjaoui
M
M
Fellmann
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D
D000818:Animals; D001345:Autoradiography; D002463:Cell Membrane Permeability; D003346:Corticotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007028:Hypothalamic Hormones; D007031:Hypothalamus / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D008543:Melanins / Q000235:genetics / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010907:Pituitary Hormones / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014667:Vasopressins / Q000235:genetics / Q000378:metabolism*
D007028:Hypothalamic Hormones; D008543:Melanins; D015345:Oligonucleotide Probes; D010907:Pituitary Hormones; D012333:RNA, Messenger; D014667:Vasopressins; C019463:melanin-concentrating hormone; D003346:Corticotropin-Releasing Hormone