I'm trying to stain spheroids embedded in alginate- gelatin hydrogel using mitochondria probe (not Immunostaining). However, I'm getting very high noise and very low signal with this dye specifically. I tried more washing with PBS for 3 times X 10 minutes. I tried also blocking with 2% FBS for 10 minutes to prevent potential non-specific binding with alginate. I tried at first using the suppliers recommendation, but the noise was high. then I tried using 50 nM and 300 nM concentrations of the dye; even though the supplier recommendation is (20 nM to 200 nM). I also tried different incubation times (30 and 60 minutes); even though the supplier recommendation is (15-45 minutes for 2D monolayers). I also tried fixation with 4% PFA; though the supplier recommends not to. It gave better results in the 2D monolayers; but it was of no help with 3D spheroids. Any suggestions?
Dear Ayman A. A.
I recommend the following articles, have a look:
1. https://www.sciencedirect.com/science/article/pii/S0014482714000263
2. Article Fully Automated One-Step Production of Functional 3D Tumor S...
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732143/
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556086/
5. https://www.frontiersin.org/articles/10.3389/fmolb.2020.00208/full
Kind Regards
Qamar Ul Islam
Have you tried low concentration of detergent to solubilize cell membranes and gain entrance inside cells? When we performed antibody staining of intracellular phenotypic expression markers we had to use detergents (Triton-X100 and CHAPS) to gain entrance to inside the cells.
Henry E Young
Thank you for your suggestion. I will try this. But do you think it will be ok with my dye? because the supplier mentions that it isn't well retained after fixation and permeabilization.
Do you make you dye yourself or you buy it pre-made? An alternative method would be to make your dye(s) using dimethyl sulfoxide (DMSO). DMSO is a universal solvent, and one of its properties is that it can traverse cellular membranes and brings entities with it into cells. I would suggest purchasing the purest form you can find.
We use 99.99% pure DMSO for cryopreservation of our stem cells with a 98% viability recovery rate. Different application, but same principle.
Do you make you dye yourself or you buy it pre-made? An alternative method would be to make your dye(s) using dimethyl sulfoxide (DMSO). DMSO is a universal solvent, and one of its properties is that it can traverse cellular membranes and brings entities with it into cells. I would suggest purchasing the purest form you can find.
We use 99.99% pure DMSO for cryopreservation of our stem cells with a 98% viability recovery rate. Different application, but same principle.
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