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Immunohistochemistry of MRD
Dr. Paul Mellor, DECVIM
2µm thick sections of formalin-fixed, paraffin wax-embedded tissues were stained for immunoglobulin α, γ and µ heavy chains, and λ and κ light chains, CD79a (monoclonal mouse anti-human CD79αcy, clone HM57, DC) and CD3 (monoclonal mouse anti-human CD3, clone F7.2.38, DC) in the presence of controls according to standard or manufacturers' techniques. Epitope retrieval 1° antibody 1° antibody dilutions 2° antibodies, visualisation system and incubation periods PK (DC) Rabbit anti-cat IgA Fc (NO) 1/1000 EnVision® (K4011 kit, DC), 60 minutes PK (DC) Rabbit anti-cat IgG Fc (NO) 1/10000 EnVision® (K4011 kit, DC), 60 minutes PK (DC) Goat anti-cat IgM Fc (NO) 1/5000 Biotinylated rabbit anti-goat Ig (E0466, DC) 45 minutes and SBC-HRP (K0377, DC) 45 minutes, DAB. MWPC Rabbit anti-human kappa light chain (A0191, DC) 1/1000 EnVision® (K4011 kit, DC), 60 minutes MWPC Rabbit anti-human lambda light chain (A0193, DC) 1/10000 EnVision® (K4011 kit, DC), 60 minutes Details of epitope retrieval, anti-Ig reagents and controls for immunohistochemical labeling of MRD.
1° antibodies were polyclonal and dilutions were made in Tris buffered saline (10mM, pH 7.5) and sections incubated for 60 minutes. The positive control was reactive cat lymph node. The negative control was mouse anti-human prostate specific antigen antibody (an unrelated antibody), applied to sections in place of the 1o antibody at a dilution of 1/1000. PK: Proteinase K was applied to sections for 5 minutes. Enzymatic proteolytic digestion was halted by application of deionised water. MWPC: Microwaveable pressure cooker (A. Menarini Diagnostics Ltd., Berkshire, UK). Samples were heated for two minutes at full pressure in sodium citrate (10mM, pH6). NO: Nordic Immunological Laboratories, Tilburg, The Netherlands. DC: DakoCytomation Ltd., Ely, UK. SBC-HRP: Streptavidin-biotin complex conjugated with horse radish peroxidase. DAB: 3,3 diaminobenzidine tetrachloride (Sigma Chemicals Co., St. Louis, MO, USA). (Mellor et al 2008). Click on an image to see a larger view
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Date Published: June 7, 2007 Paul Mellor ©
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