The tissue was incubated with primary antibodies to Na K ATPase at a dilution of 1:10 and either CA9 at a dilution of 1:1000 or VATPase at a dilution of 1:1000 in pre inc at 4 C overnight. The following day the tissue was washed approximately 10 times in pre inc for 30 minutes each at room temperature. Secondary antibodies were diluted 1:250 in pre inc and incubated with the appropriate tissue overnight at 4 C. The tissue was rinsed twice with pre inc and once with TBS at room temperature for 30 minutes each, and then mounted in 60% glycerol in TBS with phenylenediamine to diminish fluorescence quenching. In all cases, multiple primary or secondary antibodies were added simultaneously rather than sequentially. All secondary antibodies purchased were specifically ML grade . This grade is affinity purified and tested for minimal cross species immunoreactivity. For each image, an n 10 larval sections were observed. All images were captured using a Leica LSCM SP2 laser scanning confocal microscope . For each signal in each preparation, laser intensity and detector sensitivity were set to capture the full dynamic range of the fluorescence.
Quantification of signal intensity Because sources of variability exist in the imaging system as well as in preparation of the samples, we were unable to quantitatively compare signal intensity between different samples. However, we were able to compare signal between rectal cell types in the same sample. We therefore report the change in protein distribution as a change in the ratio of peak pixel intensity between the two rectal cell types in each sample. As rectal cell type mTOR signaling pathway kinase inhibitor distribution of CA and V ATPase did not change between larvae reared in freshwater and those reared in saline water, we only report the pixel intensity ratios of Na K ATPase. To quantify the immunostaining intensity between DAR and non DAR cells , the ROI function of the Leica Confocal Quantify software was used to define all cells of either type in a given tissue section.
Once we determined ATP-competitive PARP inhibitor that no pixel intensities were beyond the dynamic range of the 8 bit grey scale, the peak pixel intensity of each ROI was calculated and used as the basis of comparison between DAR and non DAR cells . Three representative recta were quantified from each group . For each rectum, the DAR non DAR peak Na K ATPase pixel intensity ratio was determined by dividing the peak pixel intensity of the DAR cells by the peak pixel intensity of the non DAR cells . The mean DAR non DAR ratios of the three recta from each group were then calculated. Finally, the mean DAR non DAR ratios of the freshwater reared larvae were plotted against the saline water reared larvae in each species using Graphpad Prism 3.0 graphing software . Standard deviation between the three recta from each group was indicated.