Planarians were treated with 2% HCl for 5 minutes on ice, and then fixed with Carnoy's fixative for 2 hours at 4°C (Umesono et al., 1997 (link)). After 1 hour wash in methanol at 4°C, planarians were bleached overnight in 6% H2O2 in methanol at room temperature. After bleaching, animals were re-hydrated in 75%, 50%, 25% methanol/PBTX (PBS with 0.3% Triton-X 100) and twice with PBTX for 5 min. Samples were then incubated in Blocking Buffer [0.6% IgG free BSA (Jackson Laboratories); 0.45% fish gelatin (Sigma, St. Louis, MO)] in PBTX for 2-4 hrs while shaking. After blocking, samples were incubated with lectins conjugated to either FITC (Con A, DBA, PNA, RCA I, SBA, UEA I, WGA, DSL, ECL, GSL II, Jacalin, LEL, STL, VVL, SNA) or Rhodamine (GSL I, LCA, PHA-E, PHA-L, PSA, Succinylated WGA, SJA) (Vector Labs) in Blocking Buffer for 4 hrs at room temperature or overnight at 4°C. Fluorescent lectin-conjugates were diluted in Blocking Buffer to concentrations ranging from 0.5-5 μg/ml. Typically, we incubated planarians with 1 μg/ml except for the following lectins that were used at 5 μg/ml: DBA, PNA, LCA, RCA I. After incubation with fluorescent lectins, animals were washed with PBTX six times for 1 hr at room temperature. DAPI (0.2 μg/ml) or TOTO-3 (0.2 μg/ml) was included in PBTX during the last wash or incubated overnight at 4°C. For double immunofluorescence and lectin staining, planarians were incubated overnight with an anti-phospho-tyrosine P-Tyr-100 antibody (Cell Signaling Technology) diluted 1:500 as previously described (Cebrià and Newmark, 2005 (link)). After washes, animals were incubated with goat anti-mouse Alexa Fluor 568 IgG secondary antibodies (Invitrogen) and WGA lectin. All samples were mounted in Vectashield (Vector Laboratories) and imaged with a Zeiss SteREO Lumar v.12 stereomicroscope, and either a CARV (BD Biosciences) or a Zeiss LSM510 confocal microscope.