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Author Topic: isolated myocytes immunostaining  (Read 4027 times)

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Offline dingding2004

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isolated myocytes immunostaining
« on: December 04, 2009, 05:15:43 PM »
Is there anybody give me a kind help?
I want to isolate myocyte from mice heart by a perfusion system,then I will do immunostaining using anbody : tni,brdu etc. Just like the photo attached.
From the photo,the myocytes is not attached on the Microscpe Slides? I think they float in the medium.
The question is:  if the myocytes is not attached on the slides,how to do the immunostaing?
I will appericate so much if anybody tell me how to do immunostaining like the photo.
Thanks in advance.


isolated myocytes immunostaining
« on: December 04, 2009, 05:15:43 PM »

Offline Cardio

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Re: isolated myocytes immunostaining
« Reply #1 on: December 05, 2009, 11:12:41 AM »
Are you sure they are not attached?

Is the whole point to label the mouse then dissociate the heart to get rid of the synthetic fibroblast contamination.

Offline dingding2004

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Re: isolated myocytes immunostaining
« Reply #2 on: December 06, 2009, 03:31:59 PM »
Are you sure they are not attached?

Is the whole point to label the mouse then dissociate the heart to get rid of the synthetic fibroblast contamination.

Thanks for answer.
To  be honest,I am not sure they are not attached.
But I think they are firstly dissociated,then labled with 2 antibodies.
How  to  to it?

More helps are needed.

Offline Cardio

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Re: isolated myocytes immunostaining
« Reply #3 on: December 10, 2009, 02:05:25 PM »
The cells are either attached or fixed.

Do you want to add drugs to the cells?

Just a generic protocol:
Collengase the heart through cannulation for 20-25 minutes.
Dissociate by pipeting the heart up and down.
Plate on Laminin plates.
Cardiomyocyes will attach faster than the fibroblast ie heavier too. 2-3 minutes.
Remove media containing fibroblast.
ITs important to use a calcium in the media.

Adjust the times as needed.
« Last Edit: December 10, 2009, 02:42:55 PM by Cardio »

Offline dingding2004

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Re: isolated myocytes immunostaining
« Reply #4 on: December 10, 2009, 04:50:54 PM »
Thanks so much.

Re: isolated myocytes immunostaining
« Reply #4 on: December 10, 2009, 04:50:54 PM »