A protocol for cryoembedding the adult guinea pig cochlea for fluorescence immunohistology

Green fluorescent protein (GFP) has been used extensively to label cells in vitro and to track them following their transplantation in vivo. During our studies using the mouse embryonic stem cell line RI B5-EGFP, we observed variable levels of fluorescence intensity of the GFP within these transfected cells. The variable fluorescence of this protein coupled with the innately autofluorescent nature of several structures within the cochlea collectively made the in vivo identification of these transplanted stem cells difficult. We have modified previously published protocols to enable the discrimination of an authentic GFP signal from autofluorescence in the adult guinea pig cochlea using fluorescence-based immunohistochemistry. The protocol described can also be used to label tissues of the cochlea using a chromogen, such as 3,3'-diaminobenzidine tetrahydrochloride (DAB). Moreover, the described method gives excellent preservation of structural morphology making the tissues useful for both morphological and quantitative studies in combination with robust immunohistochemistry in the adult guinea pig cochlea. (C) 2008 Elsevier B.V. All rights reserved