To facilitate interpretation of the expression pattern of AruRGP transcripts in starfish arm tips revealed using mRNA in situ hybridization methods, adjacent frozen sections were processed for immunohistochemical analysis using monoclonal antibody 1E11, which was generously provided by Dr. Robert D. Burke (University of Victoria, Canada; RRID AB_2617214). 1E11 is a neuron‐specific antibody to synaptotagmin B and is a marker of neural structures in echinoderms, including starfish (Burke et al., 2006 ; Saha et al., 2006 ). Importantly, the specificity of 1E11 for synaptotagmin B has been demonstrated by western blot analysis of radial nerve extracts from the sea urchin Strongylocentrotus purpuratus and comparison of immunostaining patterns observed with 1E11 and with antibodies to S. purpuratus synaptotagmin B. Further evidence of the specificity of 1E11 has been obtained by comparison of the distribution of 1E11 immunoreactivity with the distribution of synaptotagmin B mRNA in sea urchin revealed using mRNA in situ hybridization methods (Burke et al., 2006 ).
For immunohistochemistry with monoclonal antibody 1E11, starfish arm tips were lightly fixed (up to 30 minutes in 4% PFA/PBS; pH 7.4) because immunostaining with the 1E11 antibody is fixation‐sensitive (R.D. Burke, pers. commun.). Frozen sections of starfish arm tips mounted on slides were washed in PBS and then incubated for 20 minutes in PBS containing 1% hydrogen peroxide to quench endogenous peroxidases. Following washing with PBST, slides were blocked with 5% goat serum/PBST for 2 hours at room temperature. The slides were then incubated overnight at 4°C with the 1E11 antibody, diluted 1:3 with 5% goat serum/PBST. After washing with PBST, slides were then incubated for 3 hours at room temperature with goat antimouse horseradish peroxidase conjugated secondary antibodies immunoglobulins (Jackson ImmunoResearch, West Grove, PA) diluted 1:500 in PBST containing 2% goat serum. After washing in PBST, staining buffer (0.05% diaminobenzidine, 0.05% nickel chloride, 0.015% hydrogen peroxide in PBS) was applied to each slide until staining was observed. Slides were washed sequentially in PBS and autoclaved water and then coverslips were mounted using Hydromount (Natural Diagnostics). Photographs of immunostained sections and adjacent sections processed for AruRGP mRNA in situ hybridization were obtained as described above.
For immunohistochemistry with monoclonal antibody 1E11, starfish arm tips were lightly fixed (up to 30 minutes in 4% PFA/PBS; pH 7.4) because immunostaining with the 1E11 antibody is fixation‐sensitive (R.D. Burke, pers. commun.). Frozen sections of starfish arm tips mounted on slides were washed in PBS and then incubated for 20 minutes in PBS containing 1% hydrogen peroxide to quench endogenous peroxidases. Following washing with PBST, slides were blocked with 5% goat serum/PBST for 2 hours at room temperature. The slides were then incubated overnight at 4°C with the 1E11 antibody, diluted 1:3 with 5% goat serum/PBST. After washing with PBST, slides were then incubated for 3 hours at room temperature with goat antimouse horseradish peroxidase conjugated secondary antibodies immunoglobulins (Jackson ImmunoResearch, West Grove, PA) diluted 1:500 in PBST containing 2% goat serum. After washing in PBST, staining buffer (0.05% diaminobenzidine, 0.05% nickel chloride, 0.015% hydrogen peroxide in PBS) was applied to each slide until staining was observed. Slides were washed sequentially in PBS and autoclaved water and then coverslips were mounted using Hydromount (Natural Diagnostics). Photographs of immunostained sections and adjacent sections processed for AruRGP mRNA in situ hybridization were obtained as described above.
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