Assessing the potential of immunohistochemistry for systematic gene expression profiling.
Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.
Warford
A
A
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK. aw1@sanger.ac.uk
Flack
G
G
Conquer
J S
JS
Zola
H
H
McCafferty
J
J
D000071443:12E7 Antigen; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis* / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000276:immunology; D005260:Female; D005835:Genitalia / Q000737:chemistry; D006681:HLA-D Antigens / Q000032:analysis / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007422:Intestines / Q000737:chemistry; D007668:Kidney / Q000737:chemistry; D017493:Leukocyte Common Antigens / Q000032:analysis / Q000276:immunology; D008099:Liver / Q000737:chemistry; D008221:Lymphoid Tissue / Q000737:chemistry; D008297:Male; D009369:Neoplasms / Q000378:metabolism; D010179:Pancreas / Q000737:chemistry; D015203:Reproducibility of Results; D012867:Skin / Q000737:chemistry; D053668:Syndecan-1 / Q000032:analysis / Q000276:immunology; D046888:Tissue Array Analysis / Q000379:methods*
D000071443:12E7 Antigen; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; D006681:HLA-D Antigens; C508241:SDC1 protein, human; D053668:Syndecan-1; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human