I everybody, I am working with MCF-7 mammosphere cultures and I want to do some stainings. I have read papers that show very good stainings of paraffin embedded mammospheres but I have no idea how to do the embedding because spheres are too small. Is there anybody who has done this procedure before? I would appreciate a protocol for this.
Hello, Follow following steps-
1. Fix the mammosphers in 4% paraformaldehyde for 10-20 minutes.
2. Wash with PBS twice.
3. Spin at 400 rpm, remove all the pbs and resuspend in 1-1.5% Agarose (high melting point) maintained at 37 degree.
4. Allow agarose to solidify.
5. Now embed agarose in paraffin.
Hope this will help you performing the experiment.
Ciao
Thanks a lot Ravi!! seems easy, I will try. How much agarose should I add? Do I have to let it solidify in the tube or in a well?
Best
Hello, I use 100 ul Agarose for 200 spheres. After centrifugation add it the tube.
Hello Gabriela, Did it worked with you. If you have any new experiences do let me know.
Ciao
Hi Ravi, sorry for the delayed answer. The embeding worked fine but I had many problems in making the slices with the microtome that I had to try another way. What I am doing now is to embed the mammospheres without the agarose in an eppendorf tube. Practically I do everything in the small tube, all the alcohol steps, xylene and paraffin. Until now it has been working quite well.
Best!
Gaby
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