PLA₂ retrieves cytoplasmic antigens. Patterns within the dissected intestine are shown with either a combination of PLA₂ and proteinase K or PLA₂ alone using 96 hpf embryos. Desmin IHC (A) reveals a robust and reproducible pattern with a combination of PLA₂ and a low concentration of proteinase K. Both longitudinal muscle (arrow) and circular (arrow head) are observed. Smooth muscle myosin heavy chain IHC (B) shows strong staining within circular muscle (arrow) with a combination of PLA₂ and proteinase K. Both beta-catenin (C) and cadherin (E) IHC display part or all of the pattern without antigen retrieval. Addition of an intermediate concentration of PLA₂ creates an even pattern of beta-catenin (D), while high levels of PLA₂ do not result in distortion of the cadherin pattern (F). In all panels, anterior is left and posterior is right. Scale bar is 20 μM at top left corner. PLA₂, phospholipase A₂; IHC, immunohistochemistry.

PLA₂ retrieves nuclear antigens. Patterns within whole-mount embryos are revealed with PLA₂ as the sole enzyme. The pattern of proliferation is identified in 54 hpf embryos injected with BrdU (A). Repeating patterns are observed at somite boundaries (arrowheads) and at somite midline (arrow). Phospho-Smad IHC demonstrates strong nuclear staining in the midbrain (arrowhead B) and hindbrain (arrow B). There is also strong staining in the developing ear (arrows in C and D). In all panels, anterior is left and posterior is right. Scale bar is 20 μM at top left corner.

Concentrations of PLA₂ are different for nuclear and cytoplasmic serotonin antigen retrieval. High concentrations of PLA₂ are required for retrieval of nuclear serotonin within the intestine (A) and pharynx (C) but disrupt serotonin within the axons (A). Overall axon structure is not disrupted at this PLA₂ concentration as shown by acetylated tubulin IHC (B). Low concentrations of proteinase K partially permeabilize the tissue (D), while in combination with lower concentrations of PLA₂, the full pattern of nuclear (arrow) and axon (arrowhead) staining is present (E). In all panels, anterior is left and posterior is right. Scale bar is 20M at top left corner.