Developmental Expression and Functions of PLCf Figure 4. Localization of PLCf in mouse testicular spermatids, and mouse and human mature spermatozoa by immunofluores- cence. (A) PLCf (red) localized to the forming acrosome in round spermatid (top) and elongated spermatid (bottom) of testicular spreads. DNA was stained by DAPI (blue). (B, C) Mature spermatozoa were extracted from cauda epididymis and ejaculate in mouse and human, respectively. The PLCf immunofluorescence was detected in both non-permeabilized and permeabilized mouse (B) and human (C) spermatozoa. NP40 extraction abolished the detected signal on the sperm head, confirming the presence of PLCf on the surface of mature spermatozoa. Immunolocalization above was done with anti-EF with a similar result obtained with anti-hmPLCf. DNA staining is with DAPI in A (blue) and SYTOX green in B (green), DIC; differential interference contrast. Bars = 5 mm. doi:10.1371/journal.pone.0033496.g004 tissue and confirming the epididymal source of PLCf mRNA in mouse (Fig. 5A). Furthermore, we performed in situ hybridization to localize the mRNA in the epididymal epithelium. Our results confirmed the transcription of PLCf mRNA in the principal cell of the epididymal epithelium and mostly in initial segment (not shown) and caput epididymis (Fig. 5B). In order to provide evidence that PLCf mRNA is translated in the epididymis, immunocytochemistry was performed on paraffin PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33496