Overview of WMISH signals generated after pre-hybridisation treatment with NAC and/or reduction.
L. stagnalis larvae of different ages were subjected to a WMISH protocol similar to that described by Jackson et al.  (A, E, I, M and Q). This protocol was then modified by the addition of a reduction treatment (B, F, J, N and R), a NAC treatment (C, G, K, O and S) or a combination of both NAC and reduction treatment (D, H, L, P and T). Using this set of pre-hybridisation treatments, the optimal sample preparation regime for WMISH varies with respect to the target gene and the developmental stage. For engrailed and beta tubulin in younger larvae, the samples that underwent a reduction treatment display the best signal to noise ratio (B, J and N). Excess background that is revealed by reduced samples for COE and beta tubulin in older larvae (F and R) is diminished by a treatment with NAC (H and T). Black stars indicate optimal results after sample preparation involving NAC treatment and reduction. Panels A to H show larvae from a lateral perspective with the shell field oriented to the right. Larvae in I to P are viewed from dorsal and Q to T are viewed from apical.