I have intricate mouse mammary tissue organoids that have grown in 3mg/ml rat tail collagen. I would like to do immunofluorescence staining with some specific antibody. I have tried incubating the gels for upto 5 days in primary antibody and 2 days in secondary antibody. But the antibodies don't seem to penetrate through the dense collagen gel. I have also tried sectioning the gel with the vibritome and cryostat but without much success.
Any one has a bright aide about how to increase antibody accessibility?
We are talking about collagen type I, I gather.
Please, have a look at this article:
Article 3D Culture Assays of Murine Mammary Branching Morphogenesis ...
I hope this helps :)
I have read the paper, in fact followed most of the steps described in it to get the 3D structures. Whole gel staining was bad, and sectioning in cryostat with OCT led to the gel crumbling into very small pieces. So I tried the vibrotome instead, which cut the gel into small uneven sections. I am staining them right now, hopefully it will work better.
The issues you are describing may be the result of a mix of factors.
They may mean that you have used a higher concentration of rat tail collagen than necessary (collagen type I's composition is such that it provides high tensile and torsional stiffness - which would make your probes extremely difficult to process), that the temperature was off, that a step in the protocols was skipped or not followed exactly, equipment issues... who knows - it happened. Do not ponder over it for too long. Move on.
My advice (should you chose to accept it) would be - if it does not work after you have tried it a second time (most probably, there will also be tissue damage post sectioning), you can grow and harvest new material, and do it again (it should not set you back too much time-wise, considering that we are talking about mouse tissue).
If, next time around, you are uncertain as to some procedural outcomes, you can ask (judging from the nature of your research) for inter--departmental advice, in this case - the histology lab in your pathoanatomy department. They will be happy to assist you, I am sure.
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs