Hello!
I am trying to stain primary endothelial cells that have been grown on Matrigel to form tubules. I am interested in apical-basal polarity. The cells do form tubules but it is hard to image them as they are not in the same plane. The staining is also low signal and high background. I have tried removing the Matrigel and placing it on a coverslip to image under confocal; however the structural integrity of the tubules is damaged. Does anyone have experience staining and imaging endothelial tubules in Matrigel? Thank you!
My staining protocol:
1. Fix 2% PFA 20min RT
2. Block 1hr 10%normal goat serum and 3% BSA
3. Primary incubation overnight
4. Secondary incubation 1hr
5. DAPI
Dear Jiaqi
In my opinion, you should use an inverted or fluorescence microscopy with z-stack imaging (~focus stacking) capability. Although you can also take images with different focusing (different depths) and then combine them via an appropriate image processing software. Good luck
I use iBidi miu slide for angiogenesis assay, they are like microscopic slides with really small wells allowing better visualization of tube formation, it also eliminate the meniscus which could interfere with imaging, you could also try imaging at a lower magnification.
Yes, confocal is preferred but I think you could use any type of microscope since they are slides and you can visualize slides both from the back and front, you should also try optimizing your staining protocol or maybe since we are talking about immunofluorescence, you could use another endothelial marker and see if you have a better signal. You can also try taking multiple photos at different focuses, however i find it extremely hard to do a 3D reconstruction of tubes, it also requires experience with image analysis and coding.
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