Sample processing influences immunofluorescent labeling of the planarian intestine. (A) Overview of steps during fixation and processing. (B) mAb 4D2 labeling. (C) mAb 3G9. (D) mAb 2G4. For all three mAbs, labeling was best (i.e., highest signal and lowest non-specific background) in animals relaxed in magnesium and fixed with methacarn (asterisks). In some samples, magnesium treatment also improved labeling of anterior intestinal branches (arrows). “Mg”, magnesium-induced relaxation. “NAc”, N-Acetyl-L-cysteine treatment. “HCl”, HCl treatment. “FA”, formaldehyde/Triton X-100 fixation. “Mcn”, methacarn fixation. “TSA”, tyramide signal amplification. Samples were bleached in methanol/6% H2O2 (16-20h). Scale bars: 500 μm.