Fig 1: Phenotyping of three significantly different zincoproteins between non-capacitated and IVC spermatozoa.a–c CA2, d–e CCIN, and g–i CCDC39. IBFC of formaldehyde fixed and Triton X-100 permeabilized (a, d) or methanol fixed/permeabilized (g) spermatozoa; labeled with corresponding primary antibodies. Appropriate species-specific secondary antibodies conjugated to AF488 were used and coincubated with DAPI nuclear stain and PNA-AF647 for acrosome detection. Negative controls with respective non-immune sera of matching immunoglobulin concentration were performed and were published in our previous study. IBFC was performed in 5 replicates with consistent results. WB detection in individual fractions of zincoproteome purification (b, e, h), and WB detection in whole sperm extracts (c, f, i) were employed to fully characterize the capacitation changes in the studied proteins. The red arrows point to the bands of actual (CA2 ~ 32 kDa, CCIN ~ 60 kDa), and predicted (CCDC39 ~ 110 kDa) molecular weights.WB detection in sperm zincoproteome (b, e, h) and the whole sperm extracts (c, f, i) was performed in 8 replicates, and 3 replicates respectively. A statistically significant difference (P < 0.05, two-sample t-test) between non-capacitated and capacitated spermatozoa is indicated by an asterisk.