Post-fixation treatments influence immunolabeling. (A-D) Planarians labeled with mAb 3F11 after the treatments indicated at left. (E-H) Planarians labeled with mAb 2D2. (I-L) Planarians labeled with mAb 2C11. Arrows in (H) indicate the prepharyngeal region that is permeabilized by reduction treatment (compare to E). Asterisks indicate “best” treatment (i.e., highest signal intensity and specificity). Planarians were relaxed in magnesium chloride (A-D), treated with 2% HCl (A-D) or 7.5% NAc (E-L), fixed in formaldehyde/Triton X-100, and bleached in 6% H2O2/PBS (A-D) or 6% H2O2/MeOH (E-L) (12-16 hr). Reduction was conducted prior to bleaching, while proteinase K (“prot K”) and antigen retrieval (“AR”) were conducted after bleaching. Scale bars: 500 μm.