graded series of ethanol, slides were blocked with avidin- and biotin containing blocking serum followed by 10% normal goat serum [64], to avoid non-specific binding. Immunolabelling was conducted using an avidin-biotin peroxidase complex (ABC) kit (Vector Labs, Burlingame, CA). An antigen-retrieval technique was performed prior to primary antibody incubation to unmask the antigenic epitopes [46]. Slides were incubated with primary antibody overnight at 4uC, then washed in 25 mM Tris-buffered saline (TBS, pH 7.35) containing 0.1% Tween. Biotinylated goat anti-rabbit IgG was used as the secondary antibody (1:200, Vector Labs, Burlingame, CA) for 1 h at room temperature, followed by incubation with ABC. Peroxidase reaction was visualized by incubation for 10 min with 0.03% hydrogen peroxide and 0.05% diamniobenzidine tetrachloride (DAB, Sigma, St. Louis, MO). The sections were counterstained after washing with 0.1% methylene blue, dehydrated in graded ethanol, immersed in xylene, and mounted in Permount, then visualized by Nikon Eclipse E800 microscope. Immunogold labelling and electron microscopy Mouse testes were fixed in 4% formaldehyde and 0.8% gluteraldehyde and embedded in LR white (Polysciences, Inc., Warrington, PA) according to procedure described previously [46]. Tissue was then cut in ultrathin sections and mounted on Formvar-coated nickel grids (Polyscience, Inc., Warrington, PA). After blocking with 10% normal goat serum, the sections were incubated overnight at 4uC with primary antibody. Following washing, the sections were incubated with goat anti-rabbit IgG conjugated to 10 nm colloidal gold (1/20; Sigma, Mississauga, ON, Canada), and counterstained with uranyl acetate and lead citrate. The labelled sections were examined under a transmission electron microscope (Hitachi 7000). Immunofluorescence Human and mouse spermatozoa were processed and mounted on coverslips according to the previous published methods [65,66]. The sperm slides were immersed and fixed in 2% paraformalde- hyde for 40 min at room temperature, then permeabilized in 0.1 M phosphate buffered saline (PBS) with 1% Triton X-100 for another 40 min at room temperature. A proportion of each sample was kept non-permeabilized to discriminate between the sperm surface and intracellular proteins. Additionally, to address whether observed patterns of immunostaining might be associated with protein localization to acrosomal and membranous struc- tures, mouse and human samples were treated with 1% NP40 for 1 hour before fixation and processed for immunofluorescence as described above. After 25 min block in PBS- 0.1% tween containing 10% normal goat serum [64] at room temperature, the primary antibody was incubated overnight at 4uC, followed by 36 sequential washing in the labeling buffer. The second antibody mixture used to develop fluorescent reaction was TRITC- conjugated goat anti rabbit IgG (1:80 in PBST with 1%NGS; Zymed Laboratories Inc., South San Francisco, CA). Either the DNA stains SYTOX Green (0.5 mM, Invitrogen, Burlington, ON) or DAPI (1:80, Invitrogen, Burlington, ON) were mixed with the secondary antibody. The slides were mounted in an anti-fade mounting medium (VecaShield; Vector Labs, Burlingame, CA) and viewed by Zeiss AXIO Imager.A1 microscope with high numerical aperture objectives, and an AxioCam HRc camera operated by AxioVs40 V4.7 software (all Zeiss, Jena, Germany). Reverse-transcriptase PCR The mRNA expression of PLCf was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) of the mouse Developmental Expression and Functions of PLCf epididymal and testicular tissues. The RNA was extracted using Trizol as described before [67,68]. The epididymal lumen was washed with PBS several times to remove any possible contamina- tion with testicular RNA. Synthesis of cDNA and semi-quantitative RT-PCR were accomplished with specific primers targeting mouse PLCf using OneStep RT-PCR kit (Qiagen, Mississauga, ON) according to the manufacturer’s protocol with minor modifications. Reverse transcription performed in 45uC/30 min followed by initial PCR activation step in 95uC/15 min. For first PCR, the primer sequences were 59-CAAGCGGCCCAGATCATG-39 and 59- GCGTCAGTTACATGCGTCAC-39, amplifying a 1946-bp prod- uct through 40 cycles of denaturation at 94uC/10 sec, annealing at 53uC/1 min and extension at 68uC/2 min, followed by final extension at 68uC/10 min. Second PCR was performed on this product using specific primers 59-CGCAGAAGCAAGATGG- TTTTT-39 and 59-TGGGTAATCAGATGTCACAAAGG-39 am- plifying a 729 bp product through denaturation at 94uC/3 min followed by 40 cycles of denaturation at 94uC/1 min, annealing at 51uC/1 min, extension at 72uC/1 min and final extension at 72uC/ 10 min. Mouse glyceraldehyde-3-phosphate dehydrogenase testis- specific isoform (GAPDHS) was used as the housekeeping sperm specific gene [69]. The primers used for this purpose were 59- GGTCGGTGTGAACGGATTTGGC-39 and 59-GTGGGGTC- TCGCTCCTGGAAGA-39 amplifying a 234 bp product through denaturation at 94uC/3 min followed by 30 cycles of denaturation at 94uC/30 sec, annealing at 56uC/30 sec and extension at 72uC/ 40 sec and final extension of 72uC/10 min. All thermal cycling procedures were performed using PTC-100 thermal cycler (MJ Research, Watertown, MA). The PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. In situ hybridization Mouse PLCf was obtained and amplified from mouse testis by RT-PCR as mentioned above. The primers were 59-GAATTC- CATGGAAAGCCAACTT-39 and 59-GCGGCCGCTCTGAAG- TACCAAACATA-39 amplifying 1939 bp product through dena- turation at 94uC/3 min followed by 35 cycles of denaturation at 94uC/1 min, annealing at 55uC/1 min, extension at 72uC/2 min and final extension at 72uC/10 min. The cDNA was inserted into pET21b vector (Novagen, Canada) within the underlined restriction sites for EcoRI and NotI, respectively. The clone was sequenced (Eurofins MWG Operon, ON, Canada) and used as a template to amplify the approximately 300 bp product used for RNA labelling probe. The PCR was performed with 59-TTACCCTCA- CAATCCGAATCATC-39 and 59-TAATACGACTCACTATA- GGGGTTGGAAAACAGAGGAACACGA-39 primers through denaturation at 94uC/3 min followed by 30 cycles of denaturation at 94uC/1 min, annealing at 56uC/1 min, extension at 72uC/ 30 sec and final extension at 72uC/10 min. The product was purified using QIAGEN Purification Kit (Qiagen, Mississauga, ON), then served as a DNA template to synthesize DIG-labeled probe using T7 RNA polymerase and digoxigenin-11-uridine-59- triphosphate (Fermentas, Burlington, ON, Canada) according to manufacturer instructions with minor modifications. CD1 mouse epididymal tissue was fixed in 4% paraformalde- hyde, embedded in paraffin, sectioned and mounted onto poly L- lysine coated glass slides for in situ hybridization. The sections were dewaxed, rehydrated, permeabilized with proteinase K (20 mg/ml dissolved in TE: 100 mM Tris-HCl pH 7.5, 50 mM EDTA in DEPC treated water) at 37uC for 30 min, washed with PBS for 5 min, refixed with 4% paraformaldehyde for 10 min and washed twice with RNase free 26 saline-sodium citrate (SSC) buffer (300 mM NaCl, 30 mM sodium citrate, pH 7.2 in DEPC PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33496