Optimization of the immunohistochemical demonstration of keratins in paraffin wax-embedded mouse skin.
The purpose of this study was to develop an immunoperoxidase technique for the detection of cytokeratins in samples of paraffin wax-embedded adult and fetal skin from NMRI mice, with various antibodies (Troma-1, LL001, 8.60, MCK5, MCK6, AF129) that have been tested mainly on fresh-frozen sections. Each antibody was tested with three different fixatives (10% neutral buffered formalin, Bouin's fluid, and 70% ethanol) and two distinct pretreatments (enzymatic digestion with trypsin, or heat treatment). The best results, in terms of non-specific background labelling, morphological preservation and intensity of specific labelling, were obtained (1) for adult skin, by the use of Bouin's fluid, heat pretreatment and antibodies LL001, MCK5, MCK6 or AF129, and (2) for fetal skin, by the use of 70% ethanol, heat pretreatment and antibody Troma-1. Monoclonal antibody 8.60 gave the best results when the use of 70% ethanol was combined with either enzymatic digestion or heat pretreatment.
Pérez-Martínez
C
C
Department of Animal Pathology, Animal Medicine, Faculty of Veterinary Science, University of León, Spain.
García-Fernández
R A
RA
Ferreras-Estrada
M C
MC
Escudero-Diez
A
A
Espinosa-Alvarez
J
J
García-Iglesias
M J
MJ
D000818:Animals; D000911:Antibodies, Monoclonal; D005333:Fetus; D005404:Fixatives; D007124:Immunoenzyme Techniques / Q000379:methods*; D007633:Keratins / Q000032:analysis*; D051379:Mice; D016612:Paraffin Embedding; D012867:Skin / Q000737:chemistry* / Q000166:cytology; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D005404:Fixatives; D007633:Keratins