I am in search for a good technique for mounting organoid structures for immunofluorescent staining while preserving their morphology. I have tried to use double sided tape to create a ''channel''. However, with this technique the distance between the organoid samples and the microscope lens seemed to be too big causing failure to focus properly. Does anyone have good suggestions?
Use a glass-bottom Petri dish. We like the MatTek product.
https://www.mattek.com/product-category/cultureware/
Hi Anne,
I had the same issue and at the end I used commercial plates (24 WP) that are suitable for IF (cristal bottom plates), and the distance between the plate and the microscope decrease, up to 40X you can use. In this case it worked perfectly, you drop as usual your cells into the well and then in the same well you perform the IF.
These dishes are also good: https://ibidi.com/dishes/8--dish-35-mm-high-ibitreat.html
Cryosectioning is another alternative if you find penetration depth and low working distances a problem with high magnification lenses.
You can also try mounting them on a simple glass slide with 1 or 2 small cover slips (depends on the size of the organoids) on each side as spacer so the cover slip on top does not squeeze your organoids.
I tried it before and had some problems with sliding of the small cover slips on the glass slide, maybe you can fix them with super glue before mounting your organoids.
The sample can dry out with this mounting technique due to the space between glass slide and cover slip, you may use nail polish or something else for save storage.
Check this article for a detailed protocol. Hope it helps.
https://www.ncbi.nlm.nih.gov/pubmed/30548444
Generation of Pancreatic Ductal Organoids and Whole-Mount Immunostaining of Intact Organoids.
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