What steps would you need to take to develop an immunohistochemical method to identify Epstein barr virus in tonsil tissue removed from a patient.

Conducting the histochemistry assay involves a number of steps (Harlow and Lane , 1999 . First , spleens should be dissected from experimental and control mice , fixed with paraformaldehyde and then embedded in paraffin wax . Wax blocks are then trimmed and sectioned using a microtome . Alternatively , fixed tissue may be frozen and sectioned with a cryostat . Once sections are on slides , wax must be dissolved by passing slides through an organic substance such as xylene followed by rehydration through an ethanol :water series . Next the tissue is blocked , typically using bovine serum albumin or goat serum

br which aids in reducing non-specific interactions between the antibody and tissue /proteins . Following blocking , sections are incubated with the LMP1 primary antibody and then washed with Tris-buffered saline The tissue is then blocked again and treated with a secondary antibody conjugated to a fluorescent molecule . Since the LMP1 antibody was made in mice the secondary antibody must target mouse antibodies . Such secondary antibodies are readily available commercially . Following secondary antibody incubation tissue is washed again and then dehydrated , dried and mounted in an organic medium . Slides are then ready for viewing using a fluorescent or confocal microscope . These microscopes excite the fluorophore conjugated to the secondary antibody and then detect the photons of light released . The spatial pattern of fluorescence observed should directly reflect the position of the LMP1 protein since the primary antibody is bound to the protein and the fluorescing secondary antibody is bound to the primary

Once immunohistochemistry using the LMP1 antibody on transgenic LMP1 mouse tissue has shown authentic signal relative to the negative control the antibody is ready for use on human tonsil tissue as an indicator of the presence of EBV . Treatment of tonsil tissue , probing with the antibody and detection and visualization of signal would use identical procedures to those...