I am performing the AO in frozen sperm cells from wild mice. I have stained and visualized in a Zeiss fluorescence microscope with set 38 (ex 470/40, beam splitter 495 and emiss 525/50).
I only visualized green cells but as frozen samples I expected some DNA damage as background.
Should I combine this filter with another? I have the dapi filters, rhod and dic - are they helpful?
Dr. Parelho...
We have the same problem with green filter and AO in frozen semen samples of bulls and stallions... the best option to perform AO staining is with flow cytometer (although its very expensive, and is not easily avalaible)... I'm gonna search a work from Tejeda et al (1984) in which they report the use of AO and epifluorescence microscope for DNA damage evaluation in human sperm... Maybe it be helpful...
Camilo
I agree with Juan,
if possible use flow cytometry, it will help you to better discriminate the sperm population with fragmented DNA from the one with intact, double strand one. The protocol most of the researchers use is the one from Evenson et al.2002.
Good luck!
We use AO with human spermatozoa, and it works very well. It´s easier than other technique, do not need cytometry and we pubished recently that there are a good correlation with DNA fragmentation
We use a primary filter of 495 nm and a secundary filter of 500 nm. The final concentration of AO es 0,003 mg/ml and 0,001 mg/ml for etidium bromure.
I worked AO human sperm, and very well and DNA fragmentation correlates very well.
i working with Fish sperm AO giving good result and easy distinguishable.
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