Immunocytochemical localization of apolipoprotein A-I using polyclonal antibodies raised against the formaldehyde-modified antigen.
The preparation of cells for electron microscopy, in particular the fixation and embedding routine, influences the antigenicity, often resulting in a markedly reduced labelling intensity. To overcome the difficulties associated with fixation-induced changes in antigenicity, we produced antibodies against pre-fixed human apolipoprotein (apo) A-I. Purified apo A-I was fixed with 4% formaldehyde and was used to raise polyclonal antibodies in rabbits. The antiserum was purified by protein-A-Sepharose followed by affinity chromatography with the fixed antigen coupled to vinylsulphone-activated agarose. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed lipoproteins. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, indicating that the antibodies were directed against epitopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells. When HepG2 cells were exposed to anti-apo A-I antibodies followed by a secondary FITC-labelled antibody, fluorescence was found intracellularly in distinct regions. Electron microscopy revealed that the endoplasmic reticulum, and in particular the trans elements of the Golgi complexes, were the main compartments stained for apo A-I both in HepG2 cells, as shown by the immunoperoxidase technique, and in human hepatocytes, as shown by the protein-A-gold technique on ultrathin cryosections. The findings demonstrate the potential of using antibodies to fixed antigens as a strategy to overcome impaired localization due to fixation in cytochemistry at the light microscopic and electron microscopic levels.
Harrach
B
B
Department of Cell Biology, University of Münster, Germany.
Robenek
H
H
D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000032:analysis* / Q000187:drug effects / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D008075:Lipoproteins, HDL / Q000032:analysis* / Q000187:drug effects / Q000276:immunology; D008099:Liver / Q000737:chemistry*; D008297:Male; D016253:Microscopy, Immunoelectron; D011817:Rabbits
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D007106:Immune Sera; D008075:Lipoproteins, HDL; D005557:Formaldehyde