I am very interested in performing the immunofluorescence staining on a 3D spheroid model embedded in collagen type I. For example, Figure 2 A&D from the article Article Hypoxia Induces a HIF-1-Dependent Transition from Collective...
I am confused about some handling details. For example, how to transfer spheroids onto the mounting slide? How to wash the spheroid? Should I transfer the spheroid before or after the whole sample preparation process (fixation, permeation, staining)? Could anybody provide any suggestions?
Guess it depends a bit on what equipment you have available. We can do most of our imaging in the same plates we prep the spheroids. There are also special plates you can buy that will work with most conventional microscopes. For transferring to a slide, I haven't done this myself and I'm not sure how well it would work transferring the whole spheroid. Part of me thinks it will get squished when you put the coverslip on. However, I know its possible to embed your spheroid in something like agarose or matrigel and then you can have it sectioned like any other tissue with a microtome to get thin microns that you can perform immunostaining on.
Stephen Boulton Thank you for these suggestions! I also think the spheroid embedding and section is very likely to perform. And it actually reminds me that I do not need a high resolution for this immunofluorescence imaging. May I ask how do you wash these spheroids (since the condition is so different from the attached cells and the spheroid might be aspirated)?
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