I am trying to do live dead stain in sheep tibia, but the stain does not seem to go through the middle of the bone. Is there a way to do so or an alternative? I have staining with DAPI if i do it after I cut frozen sections, so I am sure there are cells.
G'day Antonio --- I'm not sure about the nature of the material you're trying to stain, it is a section, or a piece of intact bone? And which live/dead stain you are hoping to use? Can you provide a little more detail? Intrigued, Richard Horobin.
Hi Richard, I am really sorry for coming back to you so late, is just that i was really busy and forgot about posting it. I have whole tibia (7/10 um) tried cmfda and calcein am (original of cmfda) but but did not work the bone is too dense for the liquid to get through to cells, tried to section them (cryostat 7/10um thick) and tried nic translation assay but again the signal is very faint and not sure if i am doing it right
Hi Antonio,
have you tried this:
In addition, tibial bones were harvested immediately following euthanasia and 1 mm articular cartilage samples (including subchondral bone) were collected from the proximal tibia with either a tabletop bandsaw (Gryphon Corp., Sylmar, California) or low speed, precision saw (Buehler, Isomet 2000, Lake Bluff, IL), under concurrent saline irrigation to maintain hydration and control temperature. Cell viability was determined by staining with the LIVE/DEAD Viability/Cytotoxicity Kit® (L-3224, Molecular Probes, Eugene, Oregon).22 Samples were incubated in 1 ml saline with calcein AM (.5 microliters) and ethidium homodimer (10 microliters) at 37 degrees Celsius for one-half hour prior to viewing. Live cells with an intact membrane and intracellular esterase activity fluoresce bright green as calcein AM is converted to calcein. Dead, dying, or damaged cells allow ethidium homodimer to enter through a porous plasma membrane and bind to nucleic acids producing a bright red fluorescence.
it belongs to this link:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2988490/
Good luck
JRG
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