Protocol

Immunohistochemical Staining of Sectioned Tammar Wallaby (Macropus eugenii) Tissue

Department of Zoology, The University of Melbourne, Victoria 3010, Australia

¹Corresponding author (m.renfree{at}unimelb.edu.au).

INTRODUCTION

Although standard immunohistochemical protocols are used for staining tammar wallaby (Macropus eugenii) tissues, sometimes the tammar protein sequence differs slightly from that of the species against which the antibody was raised. If possible, obtain sequence for the marsupial protein to see which regions of the protein are the most highly conserved between marsupials and eutherians. When purchasing an antibody, if possible, choose one that has shown reactivity across a broad range of species, and preferably choose a polyclonal rather than a monoclonal. Because a tammar epitope may differ slightly from the one against which an antibody was raised, it may be affected differently by fixation, especially if fixatives (such as paraformaldehyde [PFA] or formalin) that cross-link proteins are used. When testing a new antibody on tammar tissues, we therefore routinely test numerous pretreatments to see which one gives optimal staining. In some cases, the optimal pretreatment differs from that suggested by the manufacturer of the antibody. This protocol describes how to use the avidin-biotin complex detection method on paraffin-embedded specimens.