Hi Everyone,
I am optimising a ex-vivo 3D culture of human tissues of +/- 300 um thickness. We want to know how many cells are alive and how many are dead at specific timepoints. I know I could do a live/dead stain and perform live imaging, but I'd rather look at the tissue morphology after fixation and sectioning. So I am looking for a live/dead stain specific for 3D tissues.
Does anyone have experience with such a staining protocol? Such a stain would need to be resistant to formaldehyde and freezing. All suggestions are welcome!
Thanks in advance.
Bram
Hi Bram
You could add Annexin-V to the cultured tissue and then fix, stain and image provided that your fixation/staining protocols do not degrade the signal. There are some tips on this Thermo page about this: https://www.thermofisher.com/uk/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis/annexin-v-staining.html
Keep in mind that this will only work for apoptotic/ dead cells. If your cells die via another mechanism such as necroptosis, this may not work. You may also need to optimise the concentration for your tissue and its thickness as I have only ever used it for cell culture.
Hope this helps!
Amy
Hi Amy,
Thanks for your suggestion! We could try to stain with Annexin V and then snap freeze the tissue in OCT, section, (fix), and image. I'll give it a try.
Best regards,
Bram
Dear Bram Piersma ,
I would suggest to add Ethidium Bromide and Hoechst33342. and snap freeze the 3D culture. The Hoechst will stain all cells and the Ethidium, since it is charged, can only pass defective membranes (dead cells). The staining should take you about 5-10 min and both compounds should be easily available in your lab or a neighbor lab. you can the even quantify everything via i.e. ImageJ.
best wishes
Sönke
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