Pre-embedding labeling methods.

Abstract

AbstractColloidal gold conjugates generally do not readily penetrate cells, even after permeabilization. Therefore, their use in pre-embedding immunostaining has been restricted to labeling cell-surface antigens for scanning (1) or transmission electron (Fig.1A) microscopy or for tracing endocytic pathways in living cells (Fig.1B). Recently, 1-nm gold conjugates that do penetrate cells and tissues much more readily have been used successfully to immunolabel intracellular structures (2,3). For pre-embedding labeling, all of the immunostaining is done prior to embedding the tissue in resin or preparing the samples for scanning electron microscopy. This method is especially useful if the antigen to be detected is sensitive to fixation. The immunostaining may be done on unfixed or lightly (4% formaldehyde) fixed samples. Following the immunolabeling, the samples may then be refixed in 2% glutaraldehyde-2% formaldehyde to give good ultrastructural preservation. Transmission electron micrograph of RBL-2H3 cells with colloidal gold conjugated to a monoclonal antibody against the IgE receptor. (A) Gold conjugate is localized primarily in coated pits. (B) Five minutes after exposing cells to antibody-coated gold, gold particles are localized in early endosomes. Bar = 0.5 μm. KeywordsBovine Serum AlbuminCacodylate BufferColloidal GoldPotassium ChlorideMagnesium ChlorideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.