Table 1.
List of primary antibodies used in this study
| Antibody | Species | Dilution | Specificity | Source | Characterization |
|---|---|---|---|---|---|
| Calretinin | mouse | 1:2000 | full-length recombinant human CR | Swant, Bellinzona, Switzerland | Nunzi et al. 2002 |
| Calretinin | rabbit | 1:5000 | full-length recombinant human CR | Swant, Bellinzona, Switzerland | Nunzi et al. 2002 |
| DGKβ | rabbit | 1 μg/ml | N-terminal region of rat DGKβ | gift of Dr. K. Goto, Yamagata University School of Medicine, Japan | Hozumi et al. 2008 |
| mGluR1α | mouse | 1:800 | fusion protein containing sequences from C-terminus of rat mGluR1α | Pharmingen | Nunzi and Mugnaini 2009 |
| mGluR1α | rabbit | 1:500 | peptide representing amino acid residues 945-1127 of mouse mGluR1α | Frontier Institute Co, Japan | Tanaka et al. 2000 |
| mGluR1α | rabbit | 1:2500 | intracellular C-terminal residues 859–1199 of mGluR1α | gift of Dr. R. Shigemoto, National Institute for Physiological Sciences, Okazaki, Japan | Shigemoto et al. 1997 |
| PLCβ1 G-12 (sc205; lot E040) | rabbit | 1:400 | peptide mapping at the C-terminus of bovine PLCβl | Santa Cruz | 1Western blot |
| PLCβ1 R-233 (sc0905; lot L008) | rabbit | 1:400 | amino acids 831-1063 mapping within an internal region of rat PLCβl | Santa Cruz | 1Western blot |
| PLCβ3 | guinea pig | 1 μg/ml | C-terminus (amino acid residues 1201-1234) of the mouse PLCβ3 | Frontier Institute Co., Japan | Nomura et al. 2007 2Western blot |
| PLCβ3 C-20 (sc403; lot J1811) | rabbit | 1:500 | peptide mapping near the C-terminus of rat PLCβ3 | Santa Cruz | 3Western blot |
| PLCβ3 H-84 (sc13958; lot C1710) | rabbit | 1:500 | amino acids1151-1234 mapping at the C-terminus of human PLCβ3 | Santa Cruz | Sarna et al. 2006 4Western blot |
| PLCβ4 | rabbit | 0.5 μg/ml | amino acids 15-74 of the mouse PLCβ4 | Dr. M. Watanabe, | Nakamura et al. 2004 |
| PLCβ4 | guinea pig | 0.5 μg/ml | amino acids 15-74 of the mouse PLCβ4 | Dr. M. Watanabe, | Nakamura et al. 2004 |
| Tbr2 | chicken | 1:400 | KHL-conjugated linear peptide corresponding to mouse Tbr2 | Millipore |
Both PLCβ1 antibodies labeled a single band at ~145-150 kDa position (see Fig. 1a).
The PLCβ3 antibody purchased from Frontier Institute recognized a single band at ~175 kDa protein but only in mouse (not shown). This antibody was previously shown to be specific for mouse brain tissue.
The PLCβ3 C-20 (sc403) recognized a single band at ~175 kDa (not shown) in both species. In rat but not in mouse this antibody intensely immunolabed mossy fibers in the granule cell layer and climbing fibers in the molecular layer (not shown). In rat the Purkinje cells showed insufficient, faint immunolabeling. In mouse the antibody immunolabed Purkinje cells, but also crossreacted with other large neurons of the brainstem (not shown).
The PLCβ3 H-84 (sc13958) labeled several nonspecific bands on the blot; particularly a band at ~250 KDa which was more intensely labeled than the 175 kDa PLCβ3 specific band (not shown). In rat and mouse brain sections the PLCβ3 H-84 antibody intensely labeled the endoplasmatic reticuli of most neurons. In mouse the Purkinje cell somata and dendrites were at best moderately labeled; we should however note, that the Purkinje cell immunolabeling pattern was comparable to the one we observed with the guinea pig anti PLCβ3 antibody (Frontier Institute). In rat the Purkinje cells were barely labeled, just above the background level. Notably, PLCβ3 H-84 was previously used to label murine cerebellar Purkinje cells (Sarna et al. 2006); the difference between our results and the previously published data might be due to differences in the antibody lots sold by the supplier.