**3. Pre and post-embedding immunogold electron microscopy of transfected HEK293T cells**

The monomeric- or hetero-oligomerization of intrinsic GPCRs cannot be ascertained by immunoelectronmicroscopic examination of brain tissues alone. Data concerning the heterooligomerization of GPCRs in brain tissues is typically acquired from three experimental phases. The first phase involves immunoelectronmicroscopic data acquired from preembedding methods and gene transfected cells. This provides important information as to whether hetero-expressed GPCRs can oligomerize or not. In other words, this method compares the expression patterns and co-localization of differently-sized immunogold

Both receptors were localized predominantly upon the cell surface and cytosolic membranes (Fig. 1. A,B). Merged images showed co-localization mainly in cell membranes (Fig. 1. C.). Our negative controls showed no positive signals in non-transfected HEK293T cells, indicating that the immunoreactivity observed in Fig. 1 was specific to the expressed

We examined the expression of A1R and P2YR in brain using using immunohistochemistry (Yoshioka *et al*., 2002, Namba *et al*., 2010). Prominent staining of A1R and P2Y2R were observed, particularly in Purkinje cells (Fig. 2A-C). Expression was predominantly restricted to cell bodies and neuronal dendrites. Importantly, co-localization of A1R and P2Y2R was observed in cell bodies within the cerebellum, but was detected within the nucleus of

Fig. 2. Confocal images of double immunofluorescence stained A1R (A; green), P2Y2R (B; red), and their merged images (C; yellow) in Purkinje cells. Mol: cerebellar molecular layer, Gr: cerebellar granule cell layer. Co-localization of A1R and P2Y2R (C; yellow) were detected in the soma of the Purkinje cells (arrows). Bar = 50 µm. Fluorescent images were collected via confocal laser scanning microscopy (Zeiss LSM410, Carl Zeiss) and each 10-µm optical slice consisted of a stack of of 20 sections (0.5-µm thick). Serial optical sections were

**3. Pre and post-embedding immunogold electron microscopy of transfected** 

The monomeric- or hetero-oligomerization of intrinsic GPCRs cannot be ascertained by immunoelectronmicroscopic examination of brain tissues alone. Data concerning the heterooligomerization of GPCRs in brain tissues is typically acquired from three experimental phases. The first phase involves immunoelectronmicroscopic data acquired from preembedding methods and gene transfected cells. This provides important information as to whether hetero-expressed GPCRs can oligomerize or not. In other words, this method compares the expression patterns and co-localization of differently-sized immunogold

recorded using an air objective lens of (40X, numerical aperture; 0.6).

receptors (data not shown).

Purkinje cells.

**HEK293T cells** 

**2.2 Results: Immunohistochemical studies in rat brain** 

particles using transfected- or non transfected-cells, yielding data that can determine the occurrence of GPCR hetero-oligomerization when using the same antibodies and immunoreactive conditions. Additionally, a particular advantage of using pre-embedding methods is that native antigenicity is maintained. The second phase is to acquire data from gene transfected cells using post-embedding methods. The reason why immunoelectronmicroscopic observation of tissues is applied with post-embedding methods is because it is difficult for a specific epitope anti-body to penetrate into the cytoplasmic region of tissue cells. Before experimenting on tissues, it is important to confirm patterns of immune-reaction with transfected cells using post-embedding methods in order to form positive controls for specific tissues. The last phase is to acquire data from tissues using post-embedding methods. It is suggested that the hetero-oligomerization of GPCRs in tissues would be very precise as an oligomer of different-sized gold particles in a given case of comparative data from single transfected GPCRs and co-transfected GPCRs.

#### **3.1 Results: Immunogold electron microscopic observations of HA-A1R and Myc-P2Y2R expressed in transfected cells**

In our laboratory, we examined the cellular localization of HA-A1R/Myc-P2Y2R in cotransfected HEK293T cells using post-embedding methods, anti-HA or anti-Myc IEM (Figs. 3A-D) (Namba *et al*., 2010). Immunogold particles were localized individually or in clusters, indicating that both HA-A1R and Myc-P2Y2R form monomers and homo-oligomers. Specificities of the gold-labeled anti-HA and anti-Myc antibodies were demonstrated by incubating A1R-transfected HEK293T cells with a mixture of both antibodies. Data showed that only A1R-labeled particles were present (Fig. 3A). No significant patterns were detected with either anti-HA and anti-Myc antibodies in mock-transfected HEK293T cells or with only secondary alone (i.e., no primary antibodies) in HA-A1R-transfected HEK293T cells (data not shown). Also, when Myc-P2Y2R-transfected HEK293T cells were incubated with both anti-HA and anti-Myc antibodies, we detected single particles (monomers) scattered all over the cells (Fig.3B). Another control for hetero-oligomerrization, HA-A1R-transfected HEK293T cells incubated with both anti-HA and Myc-P2Y2R, we observed all over the cells (Fig. 3D, inner cellular site). In HEK293T cells co-transfected with both HA-A1R and Myc-P2Y2R, clusters of different-sized particles were observed mainly at the cell surface (Fig. 3C) suggesting the formation of hetero-oligomers.

We would also like to introduce another means of investigating the cellular localization of anti-A1R/anti-P2Y1R in co-transfected HEK293T cells using post-embedding methods. Using mouse anti-A1R or rabbit anti- P2Y1R antibodies, hetero-oligomeric gold particles were clearly observed, predominantly at the cell surface (Fig.3C). This pattern concurred with patterns defined using pre-embedding methods and gold-labeled anti-HA and anti-Myc antibodies. The frequency of A1R and P2Y1R (P2Y2R) hetero-oligomers detected using post- embedding methods was smaller than that detected with pre-embedding methods using fresh specimens. This was likely to be due to polymerization occuring during the embedding process (data not shown). We consider that the native antigenicities of GPCRs in transfected cells may be reduced by polymerization treatment with LR-white, though closely-related patterns of immunoreactivity were obtained in our laboratory across differeing methods (Fig. 4). HA-A1R transfected HEK293T cells incubated with mouse anti-A1R using post-embedding methods indicated patterns (Fig. 3A, large particles) identical to those arising from pre-embedding methods (Data not shown). This data indicates that the immunoreactivety of mouse anti-A1R antibodies using LR-white post-embedding were effective in HA-A1R transfected HEK293T cells. Hetero-oligomeric gold particles of the mouse anti-A1R or rabbit anti-P2Y1R antibodies were observed at the cell surface (Fig.4B). HA-A1R-transfected HEK293T cells incubated with either mouse anti-A1R or rabbit anti-P2Y1R were also seen scattered all over the cells (Fig. 4C, cellular surface).

Fig. 3. Immunogold electron microscopy (post-embedding) method to visualise A1R and P2Y2R in transfected HEK293T cells using nanogold particles. A: Localization of HA-A1R (large particles) detected with anti-HA in HA-A1R-transfected HEK293T cells. B: Localization of Myc-P2Y2R (small particles) detected with anti-Myc in Myc-P2Y2Rtransfected HEK293T cells. C: Anti-HA and anti-Myc immuno-localization of anti-A1R and Myc-P2Y2R in co-transfected HEK293T cells. D: HA-A1R-transfected HEK293T cells incubated with both anti-HA and anti-Myc. Bars represent 100 nm.
