Igg free bsa

Planarians were equilibrated in PBSTx (5 min), then blocked O/N (16-20 hr) in BSA/fish gelatin blocking solution (0.6% IgG-free BSA (Jackson Immuno) and 0.45% fish gelatin (Sigma) in PBSTx) at RT. Supernatants were diluted 1:2 in blocking solution (i.e., one volume supernatant and one volume blocking buffer) in Figures 2 and 3, and Additional files 3, 4, and 5. In Figures 6 and 7, mAb 3F11 was diluted 1:2. In all other figures, supernatants were diluted 1:10 or 1:100 (2C11 only). Planarians were incubated O/N at 4°C. After 6-8 PBSTx washes over at least 6 hr, planarians were re-blocked for 1-2 hr at RT, then incubated with goat anti-mouse IgG + IgM HRP (Jackson Immuno) at 1:250 and DAPI (1 μg/ml) O/N at 4°C. For direct detection, goat anti-mouse IgG + IgM Dylight-488 (Jackson Immuno) was used at 1:500. After incubation in secondary antibody, animals were washed 6-8X in PBSTx over at least 6 hr, then twice in PBSTw (0.01% Tween-20 in 1X PBS) (5 min each). For TSA, planarians were incubated with FITC-Tyramide [34 (link)] at 1:1500 plus 0.005% H₂O₂ in PBSTw for 10-15 min at RT, then washed 3X in PBSTx (10 min each). Planarians were washed overnight in PBSTx, rinsed again in PBSTx, then mounted in Vectashield.

Control beads and AAL or UEA1 lectin-conjugated agarose beads were pre-blocked for 2 h in blocking buffer (2% IgG-free BSA (Jackson ImmunoResearch Laboratories)) on a rotator at 4 °C. Cells were lysed on ice in 1% Triton X-100 lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl in ddH₂O with protease and phosphatase inhibitors (Thermo Fisher Scientific)), briefly sonicated and pelleted. The resulting lysates were normalized in protein concentration to the sample with the lowest concentration, diluted to a final concentration of 0.25% Triton X-100 with dilution buffer (0% Triton X-100, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl in ddH₂O with protease and phosphatase inhibitors), incubated with 15 µl pre-blocked beads (beads were centrifuged out of a block and resuspended in dilution buffer) and rotated overnight at 4 °C. Next, the beads were washed twice with dilution buffer and subjected to (12%) SDS–PAGE and IB analysis using the indicated antibodies.

Single systems of control and ampullaectomized B. schlosseri colonies were anesthetized using MS-222 (MP Biomedicals, 103106) until oral siphons opened and were unresponsive. Systems were then fixed with 4% paraformaldyhyde (Electron Microscopy Sciences, 15710) with 0.5 M NaCl at 4°C for 12 hours. Following fixation, systems were bleached using 6% H₂0₂ in methanol under light for 1 hour at room temperature to quench auto-fluorescence. Systems were then washed through a series graded methanol and PBS with 0.1% Tween-20 (PBT) and then blocked with 5% heat inactivated horse serum (Jackson ImmunoResearch, 008-000-001) with 2 mg/ml IgG-free BSA (Jackson ImmunoResearch, 001-000-162) in PBT at room temperature for 4 hours. Systems were then incubated with either a Rabbit pan-Cadherin antibody (Cell Signaling, 4068P) (1∶500) or Rabbit pHH3 antibody (Millipore, 06-570) (1∶1000) in blocking buffer and incubated for 48 hours at 4°C. Following primary incubation, system were washed with PBT and incubated with an Alexa Fluor 488 Goat Anti-Rabbit secondary antibody (Life Technologies, A-11008) (1∶200) for 24 hours at 4°C. Systems were then washed with PBT for 6 hours at room temperature to remove unbound secondary antibody and flat mounted in Vectashield mounting medium for fluorescence with DAPI (Vector Labs, H-1200).

Cells were fixed (10 min, 4% paraformaldehyde), permeabilized (5 min; 0.5% NP40 in PBS; room temperature); blocked overnight [4°C; 3% IgG-free BSA (Jackson ImmunoResearch) and 0.02% Tween in PBS]. Cells were stained with anti-FLAG (1:2000) followed by anti-mouse Alexa Fluor 488 (1:1000), Rhodamine-conjugated phalloidin (Life Technologies, 1/4000) and DAPI (Sigma-Aldrich; 1 µg/ml), and mounted using Mowiol 4-88 (Polysciences Inc., Warrington, PA, USA).