Use of dinitrophenol-IgG conjugates to detect sparse antigens by immunogold labeling.
We describe a novel method for localizing sparse antigens in thin sections by protein A-gold labeling. The primary antibody is applied to fixed and detergent-permeabilized cells. The cells are then incubated with an anti-antibody that has been labeled with multiple dinitrophenol residues. The cells are next fixed again with glutaraldehyde and osmium tetroxide fixatives before embedding in Eponate. When thin sections are prepared, the dinitrophenol residues are readily detected with a tertiary anti-DNP antibody followed by protein A-gold labeling. This method offers good sensitivity along with superior morphology. Our test antigen for this method was the receptor for low-density lipoprotein, an antigen which had evaded detection by protein A-gold using ultra-thin cryosections.
Pathak
R K
RK
Department of Cell Biology and Anatomy, University of Texas Southwestern Medical Center, Dallas 75235.
Anderson
R G
RG
D000941:Antigens / Q000032:analysis*; D002460:Cell Line; D002478:Cells, Cultured; D003034:Coated Pits, Cell-Membrane / Q000032:analysis; D004140:Dinitrophenols; D005347:Fibroblasts / Q000032:analysis / Q000648:ultrastructure; D005404:Fixatives; D005976:Glutaral; D006046:Gold; D006801:Humans; D007074:Immunoglobulin G; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009993:Osmium Tetroxide; D011973:Receptors, LDL / Q000032:analysis*; D013205:Staphylococcal Protein A
D000941:Antigens; D004140:Dinitrophenols; D005404:Fixatives; D007074:Immunoglobulin G; D011973:Receptors, LDL; D013205:Staphylococcal Protein A; D006046:Gold; D009993:Osmium Tetroxide; D005976:Glutaral