How specific is the TUNEL reaction? An account of a histochemical study on human skin.
The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique has been described as sensitive method of labeling apoptotic nuclei in tissues, which preferentially stains apoptotic strand breaks. In the current study, three commercially available TUNEL kits for paraffin-embedded and cryostat tissues were tested to optimize this method for the studies on human skin. The investigation included normal skin (n = 10), atopic eczema (n = 4), basal cell carcinoma (n = 5), and lupus erythematosus (LE) (n = 31) sections. Additionally, the expression of certain apoptotic markers (Fas antigen and Bcl-2 protein) was studied immunohistologically on normal and LE skin. During TUNEL labeling according to the manufacturers' protocols, abnormally high background and nonspecific staining were found in all skin sections. The manipulation with the pretreatment time and, in particular, the introduction of an additional step (terminating the labeling reaction by inhibiting the terminal deoxynucleotidyl transferase activity) in two kits led to a remarkable improvement in their performance. The conclusions are that it is generally difficult to establish a functionally specific TUNEL technique for skin sections and that the choice of a kit is absolutely crucial for obtaining reliable results. Considering the extent to which the apoptosis research has been carried out recently, it is advisable to remain critical in evaluating the results. Further, it is necessary to combine the TUNEL technique with the investigation of other apoptotic markers.
Baima
Bozena
B
Department of Dermatology, University of Leipzig, Leipzig, Germany. baib@rz.uni-leipzig.de
Sticherling
Michael
M
D017209:Apoptosis; D002280:Basal Cell Carcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002467:Cell Nucleus / Q000473:pathology; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000032:analysis; D003876:Dermatitis, Atopic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods*; D008178:Lupus Erythematosus, Cutaneous / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D012867:Skin / Q000737:chemistry / Q000473:pathology*; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019014:fas Receptor / Q000378:metabolism
D004273:DNA, Neoplasm; D019253:Proto-Oncogene Proteins c-bcl-2; D011933:Reagent Kits, Diagnostic; D019014:fas Receptor