Article SND1, a NAC Domain Transcription Factor, Is a Key Regulator ...

Hi,

have you already read this articles? Here are the links:

Article Histochemical Staining of Arabidopsis thaliana Secondary Cel...

In the mentioned article the calcofluor is 0.02%. Have you tried it already?

Article A protocol for combining fluorescent proteins with histologi...

This article is explaining well the 0.1% calcofluor staining (30 min). See the section "Sequential staining" for further explaination.

Further info:

Article Exploring Cell Wall Composition and Modifications During the...

https://academic.oup.com/pcp/article/56/2/195/2755890

https://vlab.amrita.edu/?sub=3&brch=188&sim=778&cnt=1

Best

Can I ask you what's you'd like/expect to see by live-cell imaging? Would you like to follow protoxylem development over time? And if yes, for how long, at which temporal resolution? Are you specifically interested in cellulose, or is that just a way for you to visualize the cell wall?

direct red 23 (aka S4B/pontamine fast scarlet; prod. no. 212490 at Sigma Aldricht) has been used several times for in vivo staining and live-imaging of cellulose in a variety of Arabidopsis tissues.

To me, the protocols that people tend to use are rather invasive.

I've optimized a 'simple' protocol for in vivo pectin imaging in Arabidopsis roots.

I'd expect it to work for cellulose staining too, given that you find the right direct red 23 concentration.

The only thing I'm slightly worried about is the penetration of the dye to inner tissue layers. That'll be trial and error.

You can have a look at the methods in my article.

Article The auxin-regulated CrRLK1L kinase ERULUS controls cell wall...

The details are explained in the microscopy paragraph, starting with ' Pectin Ca2+-binding site oscillations were visualized...'

Good luck!

Hi Sébastjen Schoenaers ,,thanks for sharing your work. To answer your questions above : yes I want to look at secondary cell wall formation from early stage to middle and later stage while looking at the nature/behavior of actin tagged with red fluorescent protein. I am specifically interested in cellulose.

However, i'm having a hard time to stain the secondary cell wall as you say the method had to be non-invasive.

OK, in that case I would definitely try Direct Red 23.

Are you looking at the entire seedlings, or leaf, root,...?

Because that will make a big difference on how to stain your sample.

In case of root:

1) take a square petri plate

2) lay 3 glass slides on the bottom of the plate

3) fill the plate with a thin layer of growth medium, so that it just covers the slides

4) sow your plants on the medium overlaying the slides

5) grow for 6 days

6) excize your slides+medium+plants from the plate

7) cover your seedlings with ~100 µL of 0.01% direct red 23 (w/v) in liquid growth medium

you might have to play around with the concentration for your application

8) add a coverglass

9) leave to recover in a humid (plate filled with wet tissues) , 22°C environment for at least 1h

10) mount on microscope and image

Regarding your actin imaging. Do you have a line expressing an RFP-tagged actin binding protein? The RFP and direct red 23 emmission signal will overlap, which might not be ideal. It might be a better idea to use Lifeact-mVenus lines (YFP). Used it several times for in vivo imaging of actin dynamics (in the root). Works beautifully, and your signals wouldn't overlap.

Sébastjen Schoenaers I am looking at entire seedlings (5-6 days old). Yes, I tried using calcofluor to avoid signal overlap. I thought that maybe my technique in staining is the problem for not being able to view the cell wall under fluorescent microscope, not the stain (as direct red 23 can be used in a fashion similar to Calcofluor White for generic staining of cell walls in Ursache ‎et al 2018) .

I tried using 0.1% and 0.01% w/v calcofluor and incubate from 2mins to an hour, although my method of staining is different from yours as mentioned above.

I didnt add the dye in liquid medium instead i add in water. maybe i should try your method instead.

Would it be possible that the instrument i'm using is not suitable? Should i view them under confocal microscope instead of fluorescent microscope to view stained cell wall?

I would really appreciate your advice on this. Thanks

Hello, for me staining with Calcofluor White 0.01% for 30min of whole seedlings worked well.

I make a 1mL solution in an eppendorf tube and just let the seedling sit in it for at least 30mins.

It worked in a special case where I induce ectopic differentiation of all cells into protoxylem leading to a rapid chlorosis of the whole seedling, hence its death. I haven't tried it on non induced or WT seedlings though.

Here are example pictures:

Hello William Nicolas ,, I induced with DEX for 2 days to induce ectopic differentiation because i dind't want my plants to die, just enough so to visualize protoxylem on the epidermis. Great image captured btw, i hope to get the same result as you. If you dont mind, can you explain more about your method?

also what type of instrument you used to view?

Would really appreciate your help.

I would expect that even short incubation times with calcofluor or direct red 23 result in a strong labeling and fluorescence signal.

Whether you've been using water or liquid medium shouldn't matter.

It would surprise me if the staining procedure is the problem, since that's so straight forward.

As William Nicolas states, simply incubating in staining solution is sufficient to get strong labelling. I would however not recommend it if you want to keep your seedlings alive or 'non-disturbed'.

Maybe you can share some more details on your imaging setup and software settings? Microscope model, camera model, objective (magnification and numerical aperture), excitation light source and wavelength, emission filter(s), exposure time,...

Any simple fluorescence microscope with the right light source/emission filter should be able to excite and visualize calcofluor or direct red 23 staining.

Hello Aulya,

If you are using the same VND7 line, 2 days of induction should do, I saw dratic effects just by using phase contrast after one day of induction.

I induced 7do seedlings grown on MS + Gamborg B5 vits agar plates by transferring them on the same plates but supplemented with 10uM DEX.

Images were acquired with a confocal LSM 880. calcofluor white signal was excited with 405nm laser and slits set to something like 420-540nm. The seedlings were induced for 4 days here and were all white.

I also think 30min is alot, I just wanted to stain "a lot" to visualize where the phenotype is occuring.

Additionally I thought of also using Safranin. I will try it. According to this paper ( https://www.tandfonline.com/doi/abs/10.1080/10520290802373354?journalCode=ibih20), safranin has a different emission spectrum depending on the cellulose content.

Here are useful websites where you can input your staining agents and visualize the spectra. It allows you to display various filters and lasers to see if your imaging system can optimally image the staining agent:

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html

https://www.aatbio.com/spectrum/Pontamine_fast_scarlet_4B

What you can do is gather information on your microscope, if it is a confocal, it would be the lasers available and for epifluo microscopes it would be the filter cubes available.

I am sure you will get to image your seedlings!

Cheers,