Figure 2.

Specificity and validation of the anti-Na_X antibody. (A) The immune serum collected from the rabbits was assessed by ELISA and revealed the presence of the Na_X protein and not the GST tag. The microplate was coated with the Na_X peptide (1 μg/well), incubated with pre-immune serum (dotted line), or double purified immune serum (black line: antibody titer >>1/512,000). To control purification efficiency, ELISA was also performed on GST protein with double purified immune serum (dashed line). The rabbit antibodies were revealed with goat anti-rabbit IgG, HRP conjugated secondary antibody and the absorbance was read at 450 nm. (B) The purified anti-Na_X antibody (antibody concentration 1/250) was tested for immunohistochemical staining without (−) or with (+) preabsorption with the antigen peptide (50 μg/ml). Fluorescent immunostaining in the MnPO nucleus served as a positive control, whereas absence of immunostaining in the BST nucleus is a negative control. Scale bar: 200 μm (low magnification), 10 μm (high magnification). (C) The immunohistochemical staining was correlated with the presence of Na_X mRNA revealed by RT-PCR experiment carried out on micropunched region of the rat MnPO and BST. Note that the expected size of amplified products was 883 bp for Na_X and 200 bp for GAPDH (housekeeping gene). Commercial rat brain total RNA (Clontech Laboratories Inc.) was used as a positive control.