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Sensitive nucleic acid based detection methods such as in situ PCR, in situ RT-PCR and PRINS have great potential in the areas of developmental biology, pathogenesis and diagnostics. However, control of evaporation from in situ reactions is critical to ensure reliable data. Self-SealTM Reagent, a component added directly to the in situ reaction mixture, effectively controls evaporation during in situ procedures by creating an evaporation-limiting barrier around the periphery of a standard cover glass as the reaction proceeds. At the end of the procedure, the cover glass is easily removed by soaking in an aqueous solution. A model is presented for how Self-Seal Reagent controls evaporation while maintaining reagent concentrations. Self-Seal Reagent is shown to be effective in the detection of HIV sequences in cells by in situ PCR.