Etching Resin

Immunogold Labelling Protocols - No.6

Unmasking antigenic sites by etching resin (ultra-thin or semi-thin)

If the immunoreactivity is weak or non existent some attempt may be made to unmask antigens from resin embedded sections for LM or EM in order to make them more accessible to the antibodies. This masking of antigens may be due to crosslinking by the aldehyde fixation; chemical interaction with the resin; covering with other crosslinked proteins; osmication; covering of epitopes with resin, etc. When using etching techniques it is important to avoid causing further damage to the epitopes while optimally removing the resin or digesting surrounding proteins.

Methods of etching
1. For sections of tissue post fixed with osmium tetroxide, treat sections with 1-4% hydrogen peroxide solution for 5-10 min at room temperature. This is an oxidising reaction which removes reduced osmium metal and makes the sections more hydrophilic. Wash sections thoroughly in water before performing normal incubations. 2. Treat sections of osmium post fixed tissue sections with saturated sodium metaperiodate (0.1% diluted in distilled water) for 3-5 min at room temperature. This acts in a similar way to hydrogen peroxide (1). 3. Treat epoxy sections with a saturated solution of sodium hydroxide in ethanol or methanol for 30 minutes at room temperature. This removes epoxy resin from sections. Difficult to control for EM sections. 4. Treat acrylic sections with 70-100% ethanol. This partially dissolves the methacrylate bonds and removes resin slowly. Acrylics feel soapy when treated with alcohol for this reason. 5. Treat acrylic sections with weak hydrochloric acid (0.01-0.1%) for 10 min (on gold grids only). This gradually removes the resin by interfering with the ester bonds. Difficult to control without affecting the epitopes. 6. Treat sections with 0.1% trypsin in Tris buffer for 30 min at 37C. This unmasks antigens from unreactive precursors and digests surrounding epitopes. Useful for treating heavily fixed tissues (e.g. fixed in gluteraldehyde). Difficult to control selective digestion without interfering with specific epitopes.