**4. Post-embedding immunogold electron microscopy of brain tissues**

In keeping with observations gained by post-embedding methods for the study of cotransfected HEK293T cells described in Section 3.1. of this chapter, we should highlight that it is also possible to apply immunogold staining using post-embedding methods for the study of brain tissues. Comparing the dose of immunoreactivity from gold particles reflecting hetero-oligomers using co-transfected culture cells and post-embedding methods is essential in acquiring immunogold pattern data from hetero-oligomers *in situ*.

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-

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

transfected 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

In keeping with observations gained by post-embedding methods for the study of cotransfected HEK293T cells described in Section 3.1. of this chapter, we should highlight that it is also possible to apply immunogold staining using post-embedding methods for the study of brain tissues. Comparing the dose of immunoreactivity from gold particles reflecting hetero-oligomers using co-transfected culture cells and post-embedding methods

(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-P2Y2R-

**4. Post-embedding immunogold electron microscopy of brain tissues** 

is essential in acquiring immunogold pattern data from hetero-oligomers *in situ*.

incubated with both anti-HA and anti-Myc. Bars represent 100 nm.

P2Y1R were also seen scattered all over the cells (Fig. 4C, cellular surface).

Fig. 4. Immunogold electron microscopy to visualise A1R and P2Y1R in transfected HEK293T cells using nanogold particles. A: Localization of HA-A1R (small particles) detected with mouse anti- A1R in HA-A1R -transfected HEK293T cells. B: Mouse anti-A1R and rabbit anti-P2Y1R immuno-localization of HA-A1R and Myc-P2Y1R in co-transfected HEK293T cells. C: HA-A1R-transfected HEK293T cells incubated with both mouse anti-A1R and rabbit anti-P2Y1R. Bars represent 100 nm.

There are two reasons for this. Firstly, the antigenicity for the two receptor antibodies must accurately reflect hetero-oligomers or single expression. Secondly, the immunoreactivity of a particular antibody could be variable depending upon the methodology utilized, for example whether post- or pre-embedding methods were deployed. Usually, immunoreactive conditions during pre-embedding methods are much better than during post-embedding methods. However, immunoreactions involving inner tissues are technically difficult to perform. In the following section, we introduce how we can image the hetero-oligomerization of A1R and P2Y1R in brain tissues using post-embedding immunogold electron microscopy.

#### **4.1 Results: Immunogold electron microscopic observations of A1R and P2YR expressed in brain tissues**

We incubated post-embedded, primary antibody-stained rat brain tissues with two secondary antibodies labeled with gold particles (a 5-nm gold particle-conjugated goat antimouse IgG antibody for A1R, and a 10-nm gold particle-conjugated goat anti-rabbit IgG antibody for P2Y1R). As negative controls, brain tissues were stained with only secondary antibodies conjugated with different sized gold particles; no significant immunoreactivity was observed under the experimental conditions (data not shown). As found with transfected HEK293T cells (3.2-3.4), we observed clusters of different-sized gold particles at cytoplasmic membranes in cell bodies, indicating the presence of heteromeric complexes of endogenous A1R and P2Y1R in the rat cerebellum (Fig. 5). Significant immunoreactivity was

Fig. 5. Immunogold electron microscopy (post-embedding) method for the visualisation of A1R and P2Y1R in rat brain using nanogold particles. Localization of A1R (small particles) and P2Y1R (large particles) in cell surface of Purkinje cells detected with both anti-A1R and anti-P2Y1R Arrows indicate two adjacent receptors on the cell membrane. Bars represent 100 nm. CM, cell membrane; CP, cytoplasm.

particular antibody could be variable depending upon the methodology utilized, for example whether post- or pre-embedding methods were deployed. Usually, immunoreactive conditions during pre-embedding methods are much better than during post-embedding methods. However, immunoreactions involving inner tissues are technically difficult to perform. In the following section, we introduce how we can image the hetero-oligomerization of A1R and P2Y1R in brain tissues using post-embedding immunogold electron microscopy.

We incubated post-embedded, primary antibody-stained rat brain tissues with two secondary antibodies labeled with gold particles (a 5-nm gold particle-conjugated goat antimouse IgG antibody for A1R, and a 10-nm gold particle-conjugated goat anti-rabbit IgG antibody for P2Y1R). As negative controls, brain tissues were stained with only secondary antibodies conjugated with different sized gold particles; no significant immunoreactivity was observed under the experimental conditions (data not shown). As found with transfected HEK293T cells (3.2-3.4), we observed clusters of different-sized gold particles at cytoplasmic membranes in cell bodies, indicating the presence of heteromeric complexes of endogenous A1R and P2Y1R in the rat cerebellum (Fig. 5). Significant immunoreactivity was

Fig. 5. Immunogold electron microscopy (post-embedding) method for the visualisation of A1R and P2Y1R in rat brain using nanogold particles. Localization of A1R (small particles) and P2Y1R (large particles) in cell surface of Purkinje cells detected with both anti-A1R and anti-P2Y1R Arrows indicate two adjacent receptors on the cell membrane. Bars represent 100

nm. CM, cell membrane; CP, cytoplasm.

**4.1 Results: Immunogold electron microscopic observations of A1R and P2YR** 

**expressed in brain tissues** 

detected in the cell surface region (Fig. 3F). In our earlier experiments, oligomerization of A1R and P2Y2R in rat brain tissues under the same experimental conditions involved hippocampal pyramidal cells, cerebellum, and pyramidal cells in the forebrain (Namba *et al*., 2010). Hetero- and homo-oligomers of both A1R/P2Y1R and A1R/P2Y2R were detected in significant numbers at the cell surface in both transfected HEK293T cells and native brains.
