An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions.
We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.
D000096:Acetone; D000818:Animals; D002478:Cells, Cultured; D005404:Fixatives; D006046:Gold; D006514:Hepatitis B Surface Antigens / Q000032:analysis; D007120:Immunochemistry / Q000379:methods*; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D008961:Models, Structural; D009993:Osmium Tetroxide; D010084:Oxidation-Reduction; D012710:Serum Albumin, Bovine; D013997:Time Factors
D005404:Fixatives; D006514:Hepatitis B Surface Antigens; D000096:Acetone; D012710:Serum Albumin, Bovine; D006046:Gold; D009993:Osmium Tetroxide