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. 2022 Apr 19;23(9):4516. doi: 10.3390/ijms23094516

Figure 2.

Figure 2

Sequential staining of three sperm subpopulations, including sperm chromatin protamination status and global levels of sperm DNA methylation (5mC) and hydroxymethylation (5hmC). (a) An example of the staining results of three different sperm subpopulations; AB—aniline blue staining: pink (unstained) with proper chromatin protamination, purple (partially stained) with disturbed protamination, and navy blue (stained) with deprotaminated chromatin; 5mC—5-methylocytosine (primary antibody used: anti-5mC, clone 33D3, MABE146, Merck, Temecula, CA, USA; 1:200; secondary conjugated with FITC (F2012, Sigma-Aldrich, St. Louis, MO, USA; 1:400); 5hmc—5-hydroxymethylocytosine (primary antibody used: anti-5hmC, ab106918, Abcam, 1:1000; secondary conjugated with AF594 (ab150160, Abcam, Cambridge, UK; 1:800)). Microscope: Olympus BX40, CellSense Dimensions; Leica DM5500, CytoVision; magnification 1000×, oil immersion objective 100×; filters: DAPI/Triple/FITC/SpO; bar represents 6 μm. (b) An example of single measurement values generated by the software (CellSense Dimensions, Olympus).