Dear all, recently, I am immunostaining mitotic microtubule arrays of root tips in Arabidopsis. I find that the tissues alwas collapse after immunostaining, and sometimes, only epidermal and cortical cells can be immunostained clearly. Does anybody have excellent protocol? How to keep tissues intact and immunostain most of cells not only in the above two cell files but also other cell files? Could anybody give me suggestions?
here is my protocol:
1. Five-day Arabidopsis seedlings were fixed for 1 h under vacuum in 4% (w/v) paraformaldehyde in PEMT buffer (50 mM PIPES, pH 6.9, 1 mMEGTA, and 0.5 mM MgCl2, containing 0.05% [v/v] Triton X-100) at room temperature
2. Wash in the same buffer containing 0.05% Triton X-100 (PEMT) three times for 10 min each.
3. Root tips were excised on poly-l-lysine (Sigma-Aldrich)-coated slides in a droplet of water. Let samples dry at room temperature overnight.
4. rehydrated samples with PEM buffer for 10min
5.The root tips were digested with a mixture of cell wall digesting enzymes in PEM buffer with 0.4 M mannitol containing 2% [w/v] cellulase (CALBIOCHEM), 1% [w/v] pectinase, 2% [w/v] driselase (Sigma), at 37°C for 30min.
6. washed with PBS
9. The samples are treated with -20°C methanol for 10 min
10. Rehydrated in phosphate-buffered saline (PBS) for 10 min.
11. Root tips were permeabilized in PBS buffer (containing 2% IGEPAL CA-630, and 10% DMSO) for 30 min
12. Washed
13. Root tips were then incubated with 50mM Glycine in PBS (incubation buffer) for 30 min
14. Incubate in primary antibody(alpha-tubulin) in PBS containing 3% BSA at 4°C overnight.
15. Wash extensively in PBS buffer (e.g. three times 10 min).
16. Incubate with secondary antibody for 3 h at room temperature
17. Pipet DAPI solution (DAPI stock solution diluted 1:1,000 in PBS) onto the microscope slides to visualize nuclei and incubate for 30 min at room temperature.
The attachment are pictures from my immunostaining. I am really looking forward to any responses. Thakns a lot!
Suggest to look through protocols of Frantisek Baluska from Bonn or probably contact him, he is one of the very good specialists in the area.
Dear Feng,
First of all you have to admit that tissues deeper in the root, i.e. in the stele, are not hard to immunostain but they are hard to observe due to the overlying cells.
Second: I do not see any sign of "collapse" in your images, the root architecture appears brilliantly preserved.
In our hands, good imaging comes as follows: The whole procedure is carried out in 1.5ml tubes, only in the end we spread the material on microscope slides. I attach for you papers to check for yourself the whole protocol. It is simpler than yours, as a modification of that described by Sugimoto et al. 2000.
We fix like you do whole seedlings (our buffer is somewhat different, though, without triton in the fixative but with 5% DMSO), wash, digest with only macerozyme R-10 (pectinase and hemicellulase, you don't really need all thsese enzymes), cool at -20C in methanol (20min), extract with DMSO and Triton, incubate with Abs (overnight at RT), wash, DAPI if needed and then cover. The only "tricky" thing is to manipulate very gently in the tubes (sucking and dropping buffer) not to damage the roots, and to "fish" the seedlings with tweezers at the end and put them in an antifade solution. Our favourite antibody is YOL1/34 anti-alpha tubulin.
See the figures and if you are pleased, go on! If you have any question, don't hesitate to contact me.
Good luck!
Manolis (Emmanuel Panteris)
Hi, Feng, your treatments is too strong and contain too much not necessary steps. Please, use my protocol. It works very well. And I would suggest to combine microtubules with cellulose staining.
Protocol: an improved and universal procedure for whole-mount immunolocalization in plants
T Pasternak, O Tietz, K Rapp, M Begheldo, R Nitschke, B Ruperti, ...
Plant methods 11 (1), 50
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