Immunocytochemistry of renal membrane proteins on epoxy sections.
Immunocytochemistry performed on paraffin or cryosections is often hampered by poor morphology. Epoxy sections, in contrast, generally retain well-preserved tissue architecture. Immunocytochemistry, however, on epoxy-embedded sections is difficult due in part to the plastic itself and to the fixation conditions. Here, we present a technique for visualization of membrane proteins by immunocytochemistry on epoxy sections of kidneys fixed with glutaraldehyde without or with osmium post-fixation. Semithin sections were obtained from Epon 812-embedded mouse and rat kidney blocks. Before immunoperoxidase or immunofluorescence labeling, the sections were etched with the epoxy solvent, methanolic potassium hydroxide, followed by antigen retrieval using microwave heating. The sections were then treated with the primary antibody followed by secondary antibodies as usual. The distribution and expression patterns of a variety of membrane proteins, such as aquaporin (AQP)-1, AQP-2, and megalin, were identical to those observed by traditional immunocytochemical procedures on paraffin or cryosections. The advantages of our novel method include not only enhanced morphological quality but also the feasibility for investigators to visualize antigens of interest using archival specimens in Epon blocks.
Zhai
X-Y
XY
Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark.
Kristoffersen
I B
IB
Christensen
E I
EI
D000818:Animals; D000906:Antibodies; D051398:Aquaporin 1 / Q000276:immunology / Q000378:metabolism; D051399:Aquaporin 2 / Q000276:immunology / Q000378:metabolism; D004853:Epoxy Resins; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D005976:Glutaral; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000378:metabolism / Q000473:pathology*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000276:immunology / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008867:Microtomy; D009992:Osmium; D051381:Rats; D017208:Rats, Wistar; D049992:Sodium Chloride Symporters / Q000276:immunology / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*; D025262:Vacuolar Proton-Translocating ATPases / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D051399:Aquaporin 2; D004853:Epoxy Resins; D005404:Fixatives; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D008565:Membrane Proteins; D049992:Sodium Chloride Symporters; D051398:Aquaporin 1; D009992:Osmium; C004875:EPON; D025262:Vacuolar Proton-Translocating ATPases; D005976:Glutaral