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Figure 4: Immunofluorescence staining of PCNA in the Leydig cells cultured with NE and Ach on PD15. The percentage of PCNA-positive (green) cells to total Leydig cells (blue) was presented as means ± SEM. A single asterisks (*) indicates statistical significance ( 𝑃 < 0 . 0 5 ) relative to the untreated control. (a) and (b) control group cultured with the same dilution of NE and Ach. (c) and (d) NE group cultured in the presence of 10 μM NE. (e) and (f) Ach group cultured in the presence of 10 μM Ach. (b, d and f) Originated from the same photo (a), (c) and (e) with FITC staining only. Nuclei (blue) of all Leydig cells were stained with Hoechst 33258. Bar = 50 μm.