*2.7. Fluorescent Immunohistochemistry*

Paraformaldehyde fixed, and paraffin embedded boar and mouse epididymal sections were deparaffinized in xylene and hydrated through a graded series of methanol solutions. After hydration, the sections were treated to abolish autofluorescence and subjected to antigen retrieval by microwave irradiation in a 5% Urea, Tris-HCL solution, pH 9.5 [18]. Before primary antibody incubation, sections were blocked with 5% normal goa<sup>t</sup> serum (NGS) diluted with phosphate-buffered saline (PBS). The primary antibody was made in 1% NGS-PBS and incubated at 4 ◦C overnight. Slides were washed with 1% NGS-PBS before incubation with fluorescent-tagged secondary antibody and DAPI. Once completed, slides were washed in PBS before being mounted to coverslips using Vectashield mounting Media (Vector Laboratories) and sealed with clear nail polish. Images were captured at the Queen's University Cancer Research Institute Imaging Centre, using a Quorum Wave Effects spinning disc confocal microscope and analyzed using MetaMorph imaging software.

### *2.8. Mouse In Vitro Capacitation and Acrosome Exocytosis Reaction*

Spermatozoa were extracted from fresh cauda epididymis by piercing with a 26 12 gauge needle and allowing the sperm to diffuse out into a Whittens-Hepes Medium. Sperm were then incubated for 25 min at 37 ◦C, 5% CO2 in Whittens-Hepes Medium with either the inhibitor, or dimethyl sulfoxide (DMSO, Control). After treatment, sperm were diluted to a final concentration of approximately two million cells/mL with either a capacitation medium (Modified Whittens-Hepes medium supplemented with 5mg/mL BSA and 20 mM NaHCO3) or the non-capacitiation medium (Whittens-Hepes without supplementation) and left to incubate for up to 90 min at 37 ◦C, 5% CO2. For the analysis of tyrosine phosphorylation, samples were collected every 45 min, washed twice and placed in sample bu ffer before being run on a Western blot. For the acrosome exocytosis reaction, sperm cells were left to incubate in either capacitating or non-capacitating media for 60 min and then progesterone was added to a final concentration of 10 μM and left for an additional 30 min. Cells were subsequently washed in PBS and allowed to air dry on glass slides. Cells were fixed with ice cold absolute methanol for 15 min, washed in PBS and stained with PNA (15μg/mL) and DAPI for 30 min. Slides were rinsed one final time and coverslips were mounted. In each treatment, 200 cells were randomly selected and the acrosome was scored as either intact or reacting/reacted. At least three replicates of each treatment were assessed and the reported values represent the average per group. The Western blotting results were analyzed using an ANOVA with a post-hoc Tukey test, comparing the mean intensities of three replicates. The acrosome exocytosis reaction results were compared using a t-test with Welch's correction, comparing the average percentage of reacted cells from three replicates.
