TY - JOUR

T1 - A dual-immunocytochemical method to localize c-fos protein in specific neurons based on their content of neuropeptides and connectivity.

AU - Mikkelsen, J D

AU - Larsen, P J

AU - Sørensen, G G

AU - Woldbye, D

AU - Bolwig, T G

AU - Hastings, M H

AU - Ebling, F J

N1 - Keywords: Animals; Benzidines; Cricetinae; Estrus; Female; Gonadotropin-Releasing Hormone; Immunohistochemistry; Kindling, Neurologic; Male; Mesocricetus; Neural Pathways; Neurons; Neuropeptides; Phenotype; Photic Stimulation; Prosencephalon; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Sensitivity and Specificity; Suprachiasmatic Nucleus

PY - 1994

Y1 - 1994

N2 - Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 microns in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP).(ABSTRACT TRUNCATED AT 250 WORDS)

AB - Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 microns in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP).(ABSTRACT TRUNCATED AT 250 WORDS)

M3 - Journal article

C2 - 7928407

VL - 101

SP - 245

EP - 251

JO - Histochemistry

JF - Histochemistry

SN - 0301-5564

IS - 4

ER -