Demonstration of myosin heavy chain isoforms in rat and humans: the specificity of seven available monoclonal antibodies used in immunohistochemical and immunoblotting methods.
The aim of this paper was to present our experience with seven monoclonal antibodies, six of them were applied in immunohistochemistry and immunoblotting of MyHC isoforms in rats and humans, one of them, 6H1 (Lucas et al., 2000), was tested in human muscle sections only. The four antibodies specific to rat MyHC isoforms, BA-D5,SC-71, BF-35,BF-F3 (Schiaffino et al.,1989) reacted as declared both on muscle sections and immunoblots of rat except SC-71 antibody, which stained MyHC-2a and -2x bands in blots. One of the two commercially available antibodies, A4.74 antibody,reliably marked type 2a fibres of rat,but in blots it weakly stained MyHC-2a and -2x isoforms when used undiluted. The other one, F113.15F4, stained type 2a and 2x fibres and corresponding MyHC bands in blots. Therefore, using this antibody rat MyHC-2x can be additionally confirmed, which can be otherwise demonstrated only on the principle of exclusion with BF-35.Using the same set of antibodies human fast MyHC isoforms can be revealed less clearly. Namely, SC-71 and A4.74 antibodies intensively stained histochemical type 2a,predominantly expressing 2a MyHC transcripts and moderately type 2x fibres, expressing mostly 2x MyHC transcripts, in blots the antibodies recognized both fast isoforms. The 6H1antibody was the only one that selectively labelled type 2x fibres, whereas BF-35 left unstained only a variable proportion of histochemical type 2x fibres and MyHC-2x in blots. F113.15F4 did not distinguish between human fast fibre types and corresponding MyHC isoforms in blots. The negativeresults obtained with BF-F3 in muscle sections and in blots are in agreement with the absence of MyHC-2b in human skeletal muscles. Our results imply that the reactivity of antibodies specific to distinct MyHC isoforms should be carefully evaluated not only among various species but with the two different techniques used as well.
Smerdu
Vika
V
Institute of Anatomy Medical Faculty, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia. vika.smerdu@mf.uni-lj.si
Soukup
T
T
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000166:cytology / Q000276:immunology; D018995:Myosin Heavy Chains / Q000032:analysis* / Q000276:immunology; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology; D051381:Rats
D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D018995:Myosin Heavy Chains