Does anybody know a good protocol to fluorescently lable tubulin in cardiac muscle histological sections?
The best I got when imaged with a scanning confocal microscope was a fragmented network of tubulin at best with very poor quality. my protocol:
fix adults mouse heart in 4% PFA O.N. in 4°c. make paraffin blocks and prepare slides with 5µm thick sections. before staining deparaffinize and perform antigen retrieval in microwave with citrate buffer pH6. after slides are cooled to room temp, permeabilize with 0.5% triton in PBS for 10 mins, and then block with blocking for 1h (3% BSA + 3% heat-inactivated horse serum + 0.1% triton). primary antibody O.N. at 4°c (DM1A anti alpha-tubulin) and secondary for 1h at RT.
Is there a way for me to improve the protocol? or is it just not working on tissue and I should go straight to cultured cells?
All tubulins are very sensitive to the embedding temperatures. They are quickly heat-denatured when processing into paraffin, even after fixation. I process tissue as for paraffin embedding but instead embed the tissues into MBM plastic which I polymerize with a UV light at 4 degrees C. 3 and 4 um sections label very well for tubulins. A few of my papers (2013 and 2014) describe the process if you want to go this way with tissues. Otherwise, pre-embedding cells that haven't undergone the heat-denaturation temperatures will work as well.
B
sorry, missing word;
Otherwise, pre-embedding IMMUNOLABELLING of cells that haven't undergone the heat-denaturation temperatures will work as well.
There are many tubulin antibodies. The antibody you are using papers to be a mouse monoclonal. Maybe a different alpha-tubulin antibody would have a stronger specificity. It could be the reactivity between the antibody and your tissue. You could try a polyclonal first and then you can always go to a monoclonal after. The other issue as pointed out earlier, is that you could change the way you prep the tissue. Routinely we just use 3% BSA adn 0,1% Triton in PBS to block. I don't know if you need the horse serum or if that could cause any issues with labeling, likely it won't. You may be able to do a whole mount of thin tissue and avoid paraffin all together or switch to cryosectioning your tissue. There are other strategies including tissue culture that may also work for you if you are set up for that and depending on what you are doing experimentally. Either way tissue mounts or cryosectioning will make for good confocal images. I hope there is something in this answer that might help. Good luck!
Thank you for the input. I'll try cryosections next and hopefully, it will give me better results.
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