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  Indian J Med Microbiol
 

Figure 2: Subcellular localization of the active form of AMPK (phospho-Thr172-AMPK) in human spermatozoa. Phospho-Thr172-AMPK was detected in spermatozoa with the secondary antibody Alexa Fluor 488 goat anti-rabbit immunoglobulin G. Sperm were stained in green with anti-phospho-Thr172-AMPK antibody while sperm nuclei were stained with DAPI (blue). Images from (a-f): Phospho-Thr172-AMPK staining was visualized using epifluorescence (a-c), or confocal microscopy (d-f). Left panels (a and d) show bright field images, central panels (b and e) show IgG-Alexa 488 images and right panels (c and f) represent DAPI images. Images from (g-l) show negative controls: (g-i) Spermatozoa were preincubated with phospho-Thr172-AMPK specific blocking peptide (5X); (j-l) spermatozoa were incubated only with the secondary antibody Alexa Fluor 488 and primary antibody was omitted. Serial images (m-r) of confocal microscopy were obtained from a unique spermatozoon using different focus depths (5 μm thickness) and showing the overlapping IgG-Alexa 488 (green) and DAPI (blue) staining after incubation with the anti-phospho-Thr172-AMPK antibody. Images are representative of n = 6 experiments. Scale bars (5-10 μm) are indicated

Figure 2: Subcellular localization of the active form of AMPK (phospho-Thr172-AMPK) in human spermatozoa. Phospho-Thr172-AMPK was detected in spermatozoa with the secondary antibody Alexa Fluor 488 goat anti-rabbit immunoglobulin G. Sperm were stained in green with anti-phospho-Thr172-AMPK antibody while sperm nuclei were stained with DAPI (blue). Images from (a-f): Phospho-Thr172-AMPK staining was visualized using epifluorescence (a-c), or confocal microscopy (d-f). Left panels (a and d) show bright field images, central panels (b and e) show IgG-Alexa 488 images and right panels (c and f) represent DAPI images. Images from (g-l) show negative controls: (g-i) Spermatozoa were preincubated with phospho-Thr172-AMPK specific blocking peptide (5X); (j-l) spermatozoa were incubated only with the secondary antibody Alexa Fluor 488 and primary antibody was omitted. Serial images (m-r) of confocal microscopy were obtained from a unique spermatozoon using different focus depths (5 μm thickness) and showing the overlapping IgG-Alexa 488 (green) and DAPI (blue) staining after incubation with the anti-phospho-Thr172-AMPK antibody. Images are representative of <i>n</i> = 6 experiments. Scale bars (5-10 μm) are indicated