Alexa conjugated anti rabbit antibodies

Colorimetric and FISHs were performed as previously described (Pearson et al., 2009 (link); King and Newmark, 2013 (link)). Following fluorescent or NBT/BCIP development, animals were incubated with anti-acetylated-Tubulin antibody (1:1000, Cell Signaling, Danvers, MA), anti-H3P (1:1000, Millipore), or a rabbit antiserum recognized unknown epitope to visualize the lumen of PT (1:500). Primary antibodies were detected with either Alexa-conjugated anti-rabbit antibodies (1:1000; Abcam) or HRP-conjugated anti-rabbit antibodies (1:1000; Jackson ImmunoResearch). NBT/BCIP developed whole-mount in situ specimens were mounted in mounting media containing 75% glycerol and 2 M urea. Fluorescent whole-mount in situ specimens were mounted in modified ScaleA2 containing 20% glycerol, 2.5% DABCO and 4 M urea (Hama et al., 2011 (link)). For cryosectioning, fluorescently stained whole-mounted animals were fixed overnight in 4% paraformaldehyde (in PBS) at 4°C, washed three times in PBS, equilibrated in 30% sucrose, frozen in OCT, and cryosectioned (10–20 μm).

To assay ultrafiltration capacity of planarian protonephridia, 10 kDa tetramethylrhodamine-dextran (Molecular Probes, D-1817) and 500 kDa fluorescein-dextran (Molecular Probes, D-7136) at the concentration of 1 mg/ml were co-injected into the mesenchyme of the animals. After 2 hr, the animals were rinsed with an excess of 1× Montjuic salts, fixed in cold 4% paraformaldehyde (in 1× Montjuic salts), mounted in modified ScaleA2 and photographed using a Zeiss LSM-510 VIS confocal microscope. Dextran uptake was quantified by measuring the average fluorescence intensity per unit area using a standard signal to noise thresholding in Fiji (Schindelin et al., 2012 (link)). For immunostaining, after fixation, the samples were rinsed 3–4 times with PBSTx (0.3% Triton), incubated in blocking solution containing 5% horse serum in PBSTx (0.5% Triton) for 2 hr at room temperature, and then in anti-acetylated-Tubulin antibody (1:1000, Cell Signaling). Primary antibody was detected using Alexa-conjugated anti-rabbit antibodies (1:1000; Abcam).