Detection of immunoglobulin A by immunofluorescence in glycol methacrylate-embedded human colon

Several methods to demonstrate human immunoglobulin (Ig) A in glycol methacrylate (GMA)-embedded colon are explored. Following routine fixation in neutral buffered formalin, the tissue was dehydrated and embedded in GMA. Two-micron thick sections were heat-fixed (70 to 80 degrees C) to glass slides and then soaked in either xylene or acetone to etch the GMA. Following rehydration of the sections in serial alcohols and water, tissues were treated with phosphate buffered saline (PBS) and/or a variety of enzymes (trypsin, protease V, protease VI, pepsin, and papain) for varying periods of time. After an overnight soak in PBS the tissues were stained with fluorescein-conjugated anti-human IgA and then rinsed with PBS. This technique demonstrated excellent fluorescence of the IgA-containing plasma cells and of the discrete IgA-containing vesicles normally found in the surface epithelial cells of colon. Results were best with tissues etched with xylene and digested with 0.25 mg/ml protease V in PBS, pH 7.4, for 2 hr at 37 degrees C, followed by an overnight soak in PBS. Almost no fluorescence was seen in sections not digested with enzymes. The present method offers a simple convenient technique to perform immunofluorescence for immunoglobulin antigens on GMA-embedded tissue.