Epidermal cell kinetics by combining in situ hybridization and immunohistochemistry.
Double labelling can serve as a useful tool for providing information about cell kinetics in normal and hyperproliferative tissues in general, and skin in particular. We have developed a double-labelling method that combines immunohistochemistry using the monoclonal antibody MIB1 and non-isotopic in situ hybridization using either a digoxigenin-labelled RNA probe specific for histone 3 mRNA sequences or a Fluorescein-labelled oligonucleotide probe specific for histone 2b, 3, 4 mRNA sequences. Double labelling was performed on normal, tape-stripped normal skin and psoriatic skin. The three proliferation markers were also examined by single labelling. The ratio of cells in the S-phase (Ns) and the growth fraction (Ncy) was determined. In normal skin, psoriatic skin and tape-stripped normal skin after 24 h and after 48 h, we calculated that 15%, 16%, 3% and 12% of growth fraction consisted of cells in the S-phase respectively. The S-phase lasts approximately 10 h, so the cell cycle time in normal and psoriatic skin is approximately 62.5 h. At present, the MIB1/H3 digoxigenin or MIB1/H2b-H3-H4 Fluorescein double-labelling technique cannot be used routinely. Therefore, in order to understand the cell kinetic processes better, experiments are recommended to optimize these methods. From a practical point of view and for reasons of specificity and sensitivity, we prefer the Fluorescein-labelled oligonucleotide probe method.
Castelijns
F A
FA
Department of Dermatology, University Hospital Nijmegen, The Netherlands.
Ezendam
J
J
Latijnhouwers
M A
MA
Van Vlijmen-Willems
I M
IM
Zeeuwin
P L
PL
Gerritsen
M J
MJ
Van de Kerkhof
P C
PC
Van Erp
P E
PE
D002455:Cell Division; D018076:DNA, Complementary; D004076:Digoxigenin; D000078404:Epidermal Cells; D004817:Epidermis / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate; D006657:Histones / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D019394:Ki-67 Antigen / Q000032:analysis; D007700:Kinetics; D015345:Oligonucleotide Probes; D012867:Skin / Q000737:chemistry / Q000166:cytology / Q000473:pathology; D013223:Statistics as Topic
D018076:DNA, Complementary; D006657:Histones; D019394:Ki-67 Antigen; D015345:Oligonucleotide Probes; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin