**2. Immunostaining of GPCRs in transfected HEK293T cells and brain sections**

Double immunofluororescence microscopic methods is now generally employed for studying the co-localization of GPCRs in transfected cells. Transient transfection using HEK293T cells with epitope tagged-receptors (Hemagglutinin: HA- or Myc-) in expression plasmids has been performed routinely in our laboratory (Yoshioka *et al*., 2002; Nakata *et al*., 2006). Before commencing immunogold electron microscopy, we routinely analyse, the subcelluar distribution of HA-A1R and Myc-P2Y1R (P2Y2R) in co-transfected cells by immunocytochemistry and confocal laser microscopy (Yoshioka *et al*., 2001; Namba *et al*., 2010). Confocal imaging of co-localized GPCRs provides highly detailed information regarding their co-localization upon cellular organelles, an important feature for the subsequent analysis of co-localization in ultrastructural images obtained by transmission electronmicroscopy. If two genes are co-localized at specific cellular organelles, then there is

that A1R associates with P2Y1R in co-transfected HEK293T cells and in rat brain homogenates, whereby a P2Y1R agonist stimulates A1R signaling via Gi/o (Yoshioka *et al*., 2001, Yoshioka *et al*., 2002). Furthermore, in co-transfected HEK293T cells, hetero-oligomers display unique pharmacology whereby simultaneous activation of the two receptors attenuates A1R signaling via Gi/o, but synergistically enhances P2Y2R signaling via Gq/11 (Suzuki *et al*., 2006). Because A1R are widely expressed in the brain (Yoshioka *et al*., 2002), it is likely that these receptors also associate directly *in situ*; however, direct evidence of their oligomerization or precise co-localization in brain has yet to be demonstrated. In our laboratory, we are developing a new method, immunogold electron microscopic observation using different sized-immunogold particles enable visualize the oligomerization of A1R and P2Y2R (Namba *et al*., 2010). The aim of the study was to determine whether A1R and P2Y2R associate with each other in the rat brain by looking for receptor complexes with immunogold electron microscopy (IEM). This method also provides information concerning the localization and density of GPCR monomers and oligomers expressed in transfected

In this chapter, we describe both pre- and post-embedding electronmicroscopic techniques to identify cells or tissues expressing GPCRs utilizing differently-sized immunogold particles, and review IEM quantification as an efficient approach to analyze two specific types of data. One data set represents the classification of receptor formations. A1R and P2Y1R (P2Y2R) produce five receptor formations which are made up of monomers (A1R, P2YR), homo-oligomers (A1R- A1R, P2YR- P2YR) and hetero-oligomers (A1R-P2YR). The second dataset describes the estimation of receptor expression levels by counting

Establishing specific expression patterns of GPCRs at the ultrastructural level, and detecting homo- and hetero-oligomers of GPCRs in both co-transfected cultured cells and tissues, will enable us to visually understand some of the phenomena underlying signal transduction signalling pathways operating via GPCRs in a heteromeric dependent manner. It is widely accepted that drug discovery targets for rapid remedies are likely to be specific receptors expressed upon the cytoplasmic membrane. In order to establish the precise effects of new drugs, the expression patterns and expression level of A1R and P2Y1R (P2Y2R) represent significant factors to be considered, especially with regard to

**2. Immunostaining of GPCRs in transfected HEK293T cells and brain** 

Double immunofluororescence microscopic methods is now generally employed for studying the co-localization of GPCRs in transfected cells. Transient transfection using HEK293T cells with epitope tagged-receptors (Hemagglutinin: HA- or Myc-) in expression plasmids has been performed routinely in our laboratory (Yoshioka *et al*., 2002; Nakata *et al*., 2006). Before commencing immunogold electron microscopy, we routinely analyse, the subcelluar distribution of HA-A1R and Myc-P2Y1R (P2Y2R) in co-transfected cells by immunocytochemistry and confocal laser microscopy (Yoshioka *et al*., 2001; Namba *et al*., 2010). Confocal imaging of co-localized GPCRs provides highly detailed information regarding their co-localization upon cellular organelles, an important feature for the subsequent analysis of co-localization in ultrastructural images obtained by transmission electronmicroscopy. If two genes are co-localized at specific cellular organelles, then there is

cells, that are also applicable to tissues such as brain.

immunoreactive immunogold particles at the cell surface.

their association with cross-talk systems.

**sections** 

a much higher probability of hetero-oligomerization. Thus, confocal images of colocalization between A1R and P2Y1R provide an important opportunity to determine whether immunoelectronmicroscopy is possible.
