I am using protocols for immunostaining Arabidopsis MTs using tubulin antibodies. The most common protocols lead to MT labelling in the root tip and elongation zone whereas the mature zone is not. Does anyone have suggestions on how to get the mature zone to stain? I know people use freeze fracture but the stress can sometimes lead to cells losing their shape. Also any tips on getting the staining procedure to work for hypocotyls (4 dag)?
Try the wholemount protocol from Sugimoto et al., 2000
http://www.plantphysiol.org/content/124/4/1493.long
They apply only pectolyase y-23 (very good and also very expensive enzyme) enzyme digestion for 20 min and they get labeling albeit variable even in mature cells. I think that it may be safe to prolong enzyme treatment. Freeze shattering is another alternative but you will have to deal with sample losses and poor root integrity. In our laboratory we use a slightly modified version of the protocol from Sauer et al
Sauer M, Paciorek T, Benková E, Friml J. Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat Protoc. 2006;1(1):98-103
We get excellent results in root tip and growing cells (which is our major interest) but staining in more mature zones is always poor.
If possible you may try both approaches or combine them to a new protocol. I hope this helps
Hi! to improve the immunolabelling in the mature zone you can also apply cellulase in addition to pectolyase-23. I usually apply it for 30 min. Hope been helpful!!
Hi,
I still would try freeze fracture, it is rather simple and makes the interior accessible to the antibodies (with detergents of course),
Goodluck,
Tony Schaeffner
First of all apologies for not logging in and seeing this. Thank You George,Elisa and Anton. I agree that freeze shatter is helpful but i am scared the freezing step would rub the roots and lead to an artifact in the microtubule orientation. In response to George and Elisa, i figure i will start a subquestion here: Why does Cellulase improve the digestion step when used in conjunction with pectolyase, wouldn't it cause a loss in cellular integrity?? Also i have heard some people use driselase, whats your opinion/take on it??
Hello Rahul,
about cellulase, it breaks different links that pectolyase, that's why their combination helps digestion and entry of antibodies. Don't worry about the loss of cellular integrity, because amount and time is not enough to degradate all the cell wall.
About driselase, I haven't worked with it, so I can't say nothing about it.
Regards!
I've worked with the following conditions:
1% cellulase and pectolyase Y-23 in PEM buffer (50 mM PIPES, 2 mM EGTA and 2 mM MnSO4, pH 5.5), room temperature, 30 min.
Good Luck!!
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