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 R1 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.
Coleman
Bryony
B
The Department of Otolaryngology, University of Melbourne, East Melbourne 3002, Australia; The Royal Victorian Eye and Ear Hospital, East Melbourne 3002, Australia. bcoleman@bionicear.org
Rickard
Natalie A
NA
de Silva
Michelle G
MG
Shepherd
Robert K
RK
D000818:Animals; D002478:Cells, Cultured; D003051:Cochlea / Q000033:anatomy & histology / Q000378:metabolism* / Q000601:surgery; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D033581:Stem Cell Transplantation / Q000379:methods; D016610:Tissue Embedding / Q000379:methods*; D014212:Tretinoin / Q000494:pharmacology
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014212:Tretinoin