Heat-Assisted Double Indirect Immunofluorescence Staining

MICROWAVE HEAT-ASSISTED DOUBLE INDIRECT IMMUNOFLUORESCENCE STAINING 

The animal is perfused first with 1-2 ml of saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Postfixation is accomplished overnight at 4°C in the same fixative. Alternatively, the tissue can be fixed by immersion in the same fixative or 10% commercial formalin in the same buffer. 

For cryostat sectioning, the tissue specimens are cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 12 hr or until they sink to the bottom of the container. They are embedded in O.C.T compound (Miles, Elkhart, IN) and frozen in N-heptane cooled to the temperature of liquid nitrogen. Alternatively, if the antigens are resistant to paraffin embedding, the specimens can be dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. 

Sections (5 p.m thick) are hydrated and treated with 1 % bovine serum albumin or with 10% serum from the same species from which the second antibody is derived 

Antisera or monoclonal antibodies are applied by diluting as required in TBS containing 0.25% BSA (TBS-BSA). Monoclonal antibodies are applied for 1 hr at room temperature, while polyclonal antibodies are applied overnight at 4°C. 

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