Removal of mercuric chloride deposits from B5-fixed tissue will affect the performance of immunoperoxidase staining of selected antibodies.

B5 clearing is a step used before immunoperoxidase staining to remove the precipitated mercuric chloride deposits caused by B5 fixation of tissue. In the B5 clearing procedure, the slides are treated with Lugol's iodine and 5% sodium thiosulfate before antigen retrieval and the application of the primary antibody. The goal of this project was to study the effect of the B5 clearing protocol on immunoperoxidase staining on paraffin-embedded tissue, which has not been previously reported in a series of antibodies. We evaluated 75 antibodies using the 2-step clearing protocol and performed paired immunoperoxidase staining on the Ventana ES instrument, with and without the clearing protocol. We found that among 75 antibodies studied, 3 (CD5, CD30, and synaptophysin) showed total obliteration of reactivity, and 3 (ALK, Ulex, and GFAP) showed partial reduction of the staining compared with the controls. Pathologists must be aware of the possible false-negative staining effect caused by the routinely used B5 clearing protocol. Control tissues must receive the same clearing protocol (i.e., placed on case slides) to ensure detection of this effect.