Lsm 510 vis confocal microscope

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).