1.6. Immunofluorescence detection of proteins in tissue: tubulin localization in ovariole whole mounts as an example of a working protocol

One of the most widely used techniques to study the function and/or localization of proteins is known as immunolabelling or immunolocalization. This is a general technical term that defines the use of specific antibodies to identify the location of molecules or structures within cells or tissues, both in whole mounts and histological sections. Depending on the method of antibody detection, these techniques are divided into two major categories: immunofluorescence, which employs a fluorescence-conjugated secondary antibody, and immunocytochemistry, which uses an enzyme-conjugated secondary antibody and a precipitable enzyme substrate for detection. The choice of an immunolabelling protocol will take into account several factors to obtain reliable staining results, such as the specificity of the antibodies and the general conservation of cell and tissue structure.

There are currently no commercially available primary antibodies generated against honey bee proteins. For honey bee research, all antibodies are by definition heterologous ones, having been produced against a protein of another species. Depending on the honey bee protein(s) of interest, these heterologous antibodies can have good cross reactivity, as certain immunoreactive protein domains (epitopes) may be conserved. If available, it is of course preferable to use antibodies produced against specific honey bee proteins (e.g. vitellogenin).  When choosing a primary antibody, it is furthermore of interest to note whether it is a polyclonal or monoclonal one. Polyclonal ones were generated by conventional immunization of a laboratory animal, and thus are reactive with several domains (epitopes) of a certain single protein, whereas monoclonal ones were generated by hybridization and subsequent selection for a single epitope.

As far as secondary antibodies are concerned, they must, of course, be directed against the immunoglobulin type of the species in which the primary antibody was produced. Furthermore, secondary antibodies can be whole serum, purified immunoglobulins, or antibody fragments (Fab) corresponding to the antigen binding domain. All such choices will eventually depend on the question to be answered, time investment, prior laboratory experience, and availability of antibodies from commercial or non-commercial suppliers (e.g. the Developmental Studies Hybridoma Bank at the University of Iowa, http://dshb.biology.uiowa.edu, or colleagues).

As it is impractical to provide a comprehensive listing and description of all different immunolocalization techniques and their numerous variants herein, we instead describe in this section a specific protocol as an example for general guidance. This is an immunofluorescence protocol for detecting tubulins in whole-mount ovary preparations of adult honey bees. We considered this as a topic of broader interest, considering the importance of reproductive division of labour between queens and workers of A. mellifera as a colony trait. In terms of physiology and biochemistry, this involves, on the one hand, vitellogenin produced and secreted by the fat body, and on the other, the structural organization of the ovariole undergoing oogenesis. It is in the latter process where cytoskeleton proteins play a major role in the differentiation and development of the oocytes and nurse cells, both in adult bees and during postembryonic development (Schmidt-Capella and Hartfelder, 2002; Tanaka and Hartfelder, 2004; Florecki and Hartfelder, 2011).

One of the main steps in the developmental determination of the oocyte and its differentiation within a cluster of cystocytes is the presence of a microtubule-organizing centre or centrosome, which is a structure containing, amongst other proteins, all three members of the tubulin family, α- β- and γ-tubulin. Furthermore, α- and β-tubulin also form heterodimers that make up microtubules, e.g. present in the mitotic or meiotic spindle apparatus. Immunofluorescence detection of α-, β- or γ-tubulin can thus reveal several structures of interest, providing relevant information about cytoskeleton organization and organelle distribution in honey bee oogenesis.

1.6.2. Dissection and fixation of ovarioles