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In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi et al., 2012). DAB is oxidized by hydrogen peroxide in the presence of some haem-containing proteins, such as peroxidases, to generate a dark brown precipitate. This precipitate is exploited as a stain to detect the presence and distribution of hydrogen peroxide in plant cells. The protocol can be modified slightly to detect hydrogen peroxide in different types of plant tissue.

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Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves Updated version
The author made some updates (highlighted in blue) to the protocol on 09/19/2016.

Plant Science > Plant immunity > Disease bioassay
Authors: Arsalan Daudi
Arsalan DaudiAffiliation 1: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Affiliation 2: Department of Plant Pathology, University of California, Davis, CA, USA
For correspondence: aadaudi@ucdavis.edu
Bio-protocol author page: a107
 and Jose A. O’Brien
Jose A. O’BrienAffiliation: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Bio-protocol author page: a108
Vol 2, Iss 18, 9/20/2012, 29387 views, 15 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.263

[Abstract] In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi et al., 2012). DAB is oxidized by hydrogen peroxide in the presence of some haem-containing proteins, such as peroxidases, to generate a dark brown precipitate. This precipitate is exploited as a stain to detect the presence and distribution of hydrogen peroxide in plant cells. The protocol can be modified slightly to detect hydrogen peroxide in different types of plant tissue.

Keywords: Oxidative Burst, Reactive Oxygen Species, Hydrogen Peroxide, Disease Resistance, Hypersensitive Response

Materials and Reagents

  1. Arabidopsis plants
  2. DAB non-acidified powder (Sigma-Aldrich, catalog number: D8001)
  3. Tween 20 viscous liquid molecular biology grade (Sigma-Aldrich, catalog number: P9416)
  4. Sodium phosphate (Na2HPO4) electrophoresis grade (Sigma-Aldrich, catalog number: S3397)
  5. Aluminum foil
  6. HCl
  7. Na2HPO4
  8. Ethanol
  9. Acetic acid
  10. Glycerol
  11. DAB staining solution (see Recipes)
  12. 200 mM Na2HPO4 (see Recipes)
  13. Bleaching solution (see Recipes)

Equipment

  1. Arabidopsis growth chamber
  2. Magnetic stirrer and stirring bar
  3. pH meter
  4. 1 ml needless syringes
  5. 12-well microtiter plate
  6. Dessicator
  7. Shaker
  8. Water bath
  9. 50 ml Falcon tube

Procedure

  1. Preparation of DAB staining solution
    1. In 50 ml Falcon tube, add 50 mg DAB and 45 ml sterile H2O for a final 1 mg ml-1 DAB solution.
    2. Add small magnetic stirrer and reduce pH to 3.0 with 0.2 M HCl (to dissolve DAB).
    3. Cover tube with aluminium foil since DAB is light-sensitive.
    4. Add 25 μl Tween 20 (0.05% v/v) and 2.5 ml 200 mM Na2HPO4 to the stirring DAB solution.
    5. This will generate a 10 mM Na2HPO4 DAB staining solution and will pull the pH back up again.
      Note: Sometimes the DAB will still not fully dissolve, but usually very high levels of homogeneity in the solution are achieved. The DAB solution is only good for the day, made fresh.

  2. Staining leaves with DAB solution
    1. Grow Arabidopsis plants under normal conditions, or as per your requirements.
    2. Select rosette leaves on plants that are just pre-bolting (3-4 weeks typically).
    3. Apply treatment of choice, for example wounding or pathogen infiltration.
    4. As an example, we typically apply 100 μl of microbial elicitor solution dissolved in water (e.g. 0.5 μM Flg22) injected directly into the leaf via a 1 ml needleless syringe. Flg22 is a bacterial peptide epitope that is commonly used to trigger innate immune responses in plants.
    5. Ensure that similar, mature rosette leaves are selected for your treatment of choice.
    6. Sample at least 3 leaves per plant from 6 independent plants (biological replicates).
    7. It is good practice to repeat the entire experiment at least once to generate robust data.
    8. Allow plants to incubate depending on treatment. We typically leave the plants for 1 h if we are studying biotic stress responses following Flg22 infiltration for example.
    9. Sample the leaves at the desired time point by manually removing each leaf from the plant and placing in a 12-well microtiter plate. It is ok to place two or three leaves in one well.
    10. Apply 2 ml of the DAB staining solution to the leaf or leaves in the well. Adjust the volume to ensure that leaves are immersed.
      Note: It may be difficult to judge because these leaves are naturally hydrophobic, but if there is enough liquid for the total leaf tissue volume, then shaking the plate in the following steps will ensure that all parts of the leaf are in contact with the solution.
    11. Apply 2 ml of 10 mM Na2HPO4 as the control treatment for replicate leaves.
    12. Ensure that the DAB solution is taken up by the leaf by gently vacuum infiltrating the leaves. This is achieved by placing the 12-well plates in a dessicator and applying gentle vacuum for 5 min.
    13. Cover the 12-well plate with aluminium foil (since DAB is light-sensitive).
    14. Place the plate on a standard laboratory shaker for 4-5 h at 80-100 rpm shaking speed. Note: 4 h is optimum time for the DAB stain to develop for many biotic interactions where the level of hydrogen peroxide produced is quite high. It is perfectly fine to increase this incubation time if the interaction being studied produces lower levels of sustained hydrogen peroxide production. We have tried up to 8 h staining with no detrimental effects observed in the assay.
    15. Following the incubation, remove the foil and replace the DAB staining solution with bleaching solution (ethanol:acetic acid:glycerol = 3:1:1).
    16. Place the 12-well plate carefully in a boiling water bath (~90-95 °C setting on water bath is OK) for 15 min. This will bleach out the chlorophyll but leave the brown precipitate formed by the DAB reacting with the hydrogen peroxide. The time can be adjusted by ± 5 min depending on the appearance of the leaves (they should be completely devoid of chlorophyll).
    17. After 15 ± 5 min of boiling, replace the bleaching solution with fresh bleaching solution and allow to stand for 30 min. Samples at this stage can be stored at 4 °C for up to 4 days with no detrimental effects observed in our hands.
    18. Leaves can be directly visualized for DAB staining. Photographs are recommended on a plain white background under uniform lighting.

Recipes

  1. DAB staining solution (please see procedure 1)
  2. 200 mM Na2HPO4
    pH > 6.8
  3. Bleaching solution
    Ethanol:acetic acid:glycerol = 3:1:1

Acknowledgments

This protocol was adapted from Daudi et al. (2012) and Thordal-Christensen et al. (1997). We thank Jenifer Bush (Harvard Medical School) for plant growth and technical assistance, and Natalie Sykes (Royal Holloway, University of London) for assistance with DAB staining. We also thank Dr. Alessandra Devoto for excellent support and mentoring, and we dedicate this protocol to the late Professor Paul Bolwell. This work was supported by National Institutes of Health (NIH) grant R37 GM48707, National Science Foundation (NSF) grant MCB-0519898, and Biotechnology and Biological Science Research Council grant (BBSRC) BB/E021166.

References

  1. Bindschedler, L. V., Dewdney, J., Blee, K. A., Stone, J. M., Asai, T., Plotnikov, J., Denoux, C., Hayes, T., Gerrish, C., Davies, D. R., Ausubel, F. M. and Bolwell, G. P. (2006). Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47(6): 851-863.
  2. Daudi, A., Cheng, Z., O'Brien, J. A., Mammarella, N., Khan, S., Ausubel, F. M. and Bolwell, G. P. (2012). The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant Cell 24(1): 275-287.
  3. Thordal-Christensen, H., Zhang, Z., Wei, Y., Collinge, D. B. (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction. Plant J 11(6): 1187-94.


How to cite this protocol: Daudi, A. and O’Brien, J. A. (2012). Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves. Bio-protocol 2(18): e263. DOI: 10.21769/BioProtoc.263; Full Text



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7/23/2015 7:42:00 AM  

Wenlu Bi
NWAFU

Hi, Arsalan
Thanks for your protocol.
But I have two questions.
1. If i want to make a 1 L DAB solution, how much water could be used? if i could added water to the powder until the volume is 1 liter?
2. About the bleaching solution. what do you mean by Ethanol: Acetic Acid: Glycerol=3:1:1 ? Does it mean that i should put 3 portion ethanol, 1 portion acetic acid and 1 portion glycerol in 5 portion water?

many thanks if you could answer me!

BTW, i am doing the experiment. I would post the result if i got good one.

7/23/2015 8:13:33 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Hi Wenlu,
Thanks for your message.

To make 1 L DAB solution, I would recommend adding 900 ml sterile distilled water to 1 g DAB, then add proportional Tween-20 and top up with 200 mM Na2HPO4. Also, try to use acidified DAB instead of just DAB, then you don't need to do the acidification step in my protocol.

Yes, Ethanol: Acetic Acid: Glycerol=3:1:1 means 3 portion ethanol, 1 portion so on. Don't make it in water. Take 300 ml Ethanol, add 100 ml Acetic acid and 100 ml glycerol, and use this as the bleaching solution.

It would be great if you post your result here. Good luck!

7/23/2015 8:45:22 AM  

Wenlu Bi
NWAFU

Thanks a lot for your answer. It is very helpful. I will try again as you recommend !

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11/2/2014 11:52:15 AM  

KELLY JOHANNA AVILA MENDEZ
UNIVERSIDAD NACIONAL DE COLOMBIA

Dear Daudi

My name is Kelly Avila. I´m doing my PhD in Biotechnology and my thesis is related to resistance genes in oil palm in response to P. palmivora.

I'm interested in make a staining DAB for evaluating ROS response in my pathosystem.

I´ve never done this kind of protocol, when I found the protocol that you sharing in http://www.bio-protocol.org/e263 I feel confidence to make it. It´s very well explained.

But I have a doubts and I would like to know if you can give me any advice.

Due to the leaf size (leaf of oil palm clones), I would have to use a lot of quantity of DAB. Taking into account that this kind of protocol is for detecting ROS it is well if I make the staing with leaf discs (in orden to safe DAb reactive), or only works when is used in complete leaf in order to avoid mechanical stress when cut the discs.

Any advice would help me a lot for my job

Thank you very much and hope I have not bothered with my request

11/10/2014 11:52:04 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Hi Kelly, thanks for your message. Hope I can help. Using leaf discs is actually a more common way to do DAB (and most types of ROS) staining. I just did whole leaves because it is easy with Arabidopsis as they're small, similar sized and easy to fit into 12 and 6 well plates. I would encourage you to use leaf discs for you oil palm leaves. The bigger the better, so 0.5 cm diameter minimum I would suggest as I guess your leaves are quite big. There will be mechanical stress when you cut the leaves (ideally with a cork borer), but the way to overcome this is to allow your leaf discs to equilibrate and stabilize in sterile water for about 12 - 24 hours (without any agitation). Then you can proceed to do the DAB assay on those discs as described above. Also make a note of the new DAB I recommend to use (in one of the comments above). Please update us with your results and how it worked in your hands (on this same Q&A thread). Good luck!!

3/4/2015 9:12:24 AM  

KELLY JOHANNA AVILA MENDEZ
UNIVERSIDAD NACIONAL DE COLOMBIA

Dear Daudi,


I'm still working with this protocol. But I had problems with the results, I think that these results is for technical details.
But I wondering why in DAB protocols is don´t required extra H2O2 like other protocols.
I would like to confirm (maybe I´m confused with the chemistry of reaction).
If DAB is oxided by H2O2, the ending of the test is quantify indirectly H2O2 production, and I can assume that brown coloration is in response to pathogen attack, so all brown stains are produced by the pathogen presence

3/16/2015 4:18:19 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Hi Kelly, you are right about the chemistry: DAB is oxidized by hydrogen peroxide, which in turn leads to the dark brown precipitate/color you see. Hydrogen peroxide is not necessarily only produced by pathogen attack (i.e. the oxidative burst) - This is where you may be having a problem. ROS is produced due to all kinds of stresses on plant tissue - so if your samples are stressed for any reason, there may be background ROS (and hence the brown stain). The key is to minimize any other stress so the hydrogen peroxide produced by the pathogen attack is relatively higher than basal levels, and this difference is observed by staining. Does this make sense?

4/28/2015 6:46:21 AM  

KELLY JOHANNA AVILA MENDEZ
UNIVERSIDAD NACIONAL DE COLOMBIA

Hi Arsalan

Thanks a lot for yor your help.

Taking account all your advices and the other questions. I wondering if is better quantify intensity of staining or just express as positive or negative. Because the stain is not uniform. I tried with different programs but quantifiy this kind of stains is difficult. Any suggestion?

5/4/2015 7:01:51 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Hi Kelly,
Thanks for your message. Could you share some representative images of your leaves using the "Reproducibility Feedback" feature above? That way I can see how the staining is spread across your leaves and advise you on the best programs/tools for quantifying the stain.

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9/27/2014 11:43:47 AM  

Tommy Phannareth
University of Maryland

When I add in the base, the DAB solution develops a white precipitate, is this normal?

9/28/2014 6:27:54 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

I wouldn't expect a white precipitate. Could something be precipitating in the buffer from before? Incomplete acidifying of the DAB has led to light brown precipitation in our hands. If that is what you're seeing, a low level of that should not affect the experiment. In addition to this, I have posted an update in the comments above. After publishing this protocol, I realized that using the DAB tetrachloride salt prevents the need for the acidification step. With this, I never saw any DAB precipitation again. Such search for 3,3'-Diaminobenzidine tetrahydrochloride (the one by Santa Cruz sc-209686 works fine - I'm quite sure the one from Sigma will be fine too).

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2/23/2014 11:37:16 PM  

King cougar
ECUST

I Want to know that this protocol can stain Arabidopsis roots?

2/27/2014 5:13:35 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

The DAB stain has been used by several other researchers to stain Arabidopsis root tissue. My protocol however is designed for leaf tissue; hence the destain step. Since roots do not have chlorophyll, in principle you can modify this protocol but remove the destain step.

3/1/2014 7:23:13 PM  

King cougar
ECUST

Thank you. I really admire your enthusiasm.

10/20/2015 1:53:10 AM  

Joice Wang
Aromalake Chemical

We are manufacturer of 3,3'-Diaminobenzidine(DAB) in China.
We provide qualified DAB with low price.
Anyone interested can contact us:
http://www.aromalake.com
Thank you!

The latest modification time: 10/20/2015 1:54:50 AM

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2/9/2014 4:41:48 AM  

prashant soni
DOR

can you explain how to handle the Imagej software with light microscopic Image(without scale)
or is it possible to take help with cellprofiler here, kindly explain me in detail.

2/10/2014 3:59:30 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Hi Prashant, the principle behind using ImageJ or CellProfiler is essentially the same. Since I prefer CellProfiler, I would recommend trying with that (http://www.cellprofiler.org/). You will notice that they have extensive tutorials and FAQs which can be applied to any type of image analysis, not just ROS staining with DAB. Briefly, CellProfiler has a tool with which you can manually identify regions of interest on your leaf. In the case of DAB, you would use pixel intensity. Over a certain pixel intensity threshold, you would set the identification as "DAB stain". Next, you can feed as many images as you like into that pipeline and it will sort based on the threshold you set. With this, you can get a quantitative assessment of the amount of staining in your tissue. Please let me know if this helped answer your question.

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4/15/2013 8:45:29 AM  

Ren Bing
Northwest A & F University

I have followed the Thordal-Christensen's method without using Tween 20 and Na2HPO4 to detect the H2O2 in apple leaves infected by pathogen,but it doesnot work.I want to know the reason that you add Tween 20 and Na2HPO4 to DAB solution. Thank you!

4/16/2013 11:22:33 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Tween 20 aids in the even dispersal and uniform infiltration of DAB. A precisely buffered and pH maintained DAB solution maintains its stability for longer during the assay. Both these features contribute to the robustness and reproducibility of this protocol.

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2/13/2013 7:46:25 PM  

I have followed your protocol and it has worked but the staining is a bit faint(after infiltration incubated in DAB for 4 hrs )Should I increase incubation time in DAB by how much?

2/19/2013 12:36:47 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

I am glad to hear that our protocol worked in your hands. Yes, incubating longer (5 - 8 hours) is OK. Did you de-stain efficiently? Did you ensure that the vacuum infiltration was correctly carried out and the vacuum pressure was relatively strong (this will enable the stain to be taken up by the leaves)? Was your DAB prepared fresh and was the DAB staining solution quite homogenous? It is difficult to precisely answer your question as several aspects can impact the strength of your stain, but if you keep all these points (and of course several others in your experimental design), then hopefully you'll be able to see a stronger stain. Does your positive control definitely expect higher stain / ROS production?

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12/25/2012 5:38:45 AM  

How to determine the concentration of hydrogen peroxide , by the color of stains or the number of stains? Thank you!

1/10/2013 9:56:27 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Both the intensity and distribution of the stain can be used to determine the extent of DAB staining which is consequently proportional to hydrogen peroxide accumulation levels. Both of these can be worked out using a variety of bioimaging softwares available such as ImageJ or CellProfiler. A bit of effort is required to set up these methods and the best advice I can give is reading the tutorials on these software to learn their capability, then playing around with high resolution photographs of your DAB stains. However, DAB is not a specifically quantitative stain in the protocol that I have described, and I do not know of a way to quantify the actual, accurate concentration of H2O2 with our methods. For that, you can use other methods such as xylenol orange or luminol detection.

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12/17/2012 5:31:33 AM  

How to apply gentle vacuum for 5 min.Other steps can be used to replace it.Thank you!

12/22/2012 3:57:01 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Using a vacuum pump.

I have not tried any other method.

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12/16/2012 11:16:38 PM  

Using the syringe to inject leaves, will the leaves be wounded, and show dark brown after staining? if so, it's hard to tell whether the H2O2 production is caused by elicitors or wounding, for example.
I have done the experiment once, and the infiltration site showed dark brown. How to solve this problem? Thank you!

12/22/2012 3:56:25 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

This is where experimental skill comes in. It took me and several other members of the lab, as well as a large number of collaborators and additional colleagues many, many attempts to get it right. The thing to get right is not to wound the leaf, which can happen with "heavy hands" or mishandling. It is very common with students and first-timers. Over time, a good experimenter will learn the amount of pressure that is required to correctly infiltrate the leaf tissue. Applying water 30 minutes below on the leaf surface that you will be infiltrating has helped as well. It enables the liquid to be dispersed more easily into the apoplast. What is important to keep in mind is NOT to force the liquid in, as this will damage the leaf surface. With practise and correct positioning of the syringe, you will slowly learn that with the right gentle finger pressure, the liquid actually fills very passively and easily into the tissue.

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12/9/2012 12:47:08 AM  

The blade has a certain thickness, can be directly observed with a microscope.how to photographs on a plain white background under uniform lighting.Thank you!

12/22/2012 3:49:21 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Depends on what you want to look at. If you want an overview of the distribution and overall intensity of the DAB stain, then just place the de-stained leaf on a plain white background under uniform lighting and you should get a clear image of the stain.

If you want to look at it under the microscope, just mount the leaf as you would normally on a glass slide and view under a light microscope. As the stain requires no phase contrast or fluorescence detection, it is simple to adjust the plane according to what cells you wish to see.

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12/7/2012 12:54:23 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Protocol update: It is possible to buy a DAB tetrachloride salt, which enables it to dissolve much better in aaqeous solutions. Using this version of DAB, it is possible to avoid the acidifying step.

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12/6/2012 7:29:54 PM  

How to place leaves in a 12-well microtiter plate

12/7/2012 12:53:15 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

Well, it depends on the size of your leaf. For Arabidopsis, rosette leaves are small enough to fit quite happily in 12-well plates (for most lines I've worked with). Ofcourse, the simplest solution is to switch to a 6-well plate if your leaves are bigger. This will not negatively influence the protocol. Another reasonable option is to make leaf discs or leaf cuts. I have tried with both, and the protocol works perfectly fine in both. The thing to consider here is, please float your leaf discs or cuts for at least 8 hours in water prior to starting the DAB staining experiment, because this will stabilize the levels of ROS that would be induced due to wounding when you make your leaf disc or cut. It is important to stabilize this. I have tried this many times and it works fine.

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12/6/2012 6:55:28 PM  

I want to know if this method can be apply to apple leaves. thank you!

12/7/2012 12:49:36 PM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

There is no reason why not. I have not tried with Apple leaves. This protocol would definitely need to be modified based on your tissue though, and they're several places where it can be modified if you think about it in a smart way. For e.g. rice and wheat leaves are tougher than Arabidopsis, thicker, with more waxy cuticle etc. Therefore, you need to ensure the vacuum infiltration is effective. This is how the leaf tissue takes up the stain. For e.g. I modified that part of the protocol to use a tougher/stronger vacuum pump. You can also play around with the duration of staining and de-staining. There are additional ways to de-stain a leaf, you can boil for longer, you can change the de-stain solution more times etc. So, in summary, yes you should be able to adapt this protocol for apple leaves. Please let me know if it works, I'd be curious to hear of your outcome.

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11/9/2012 12:59:30 AM  

1ml needless syringes ,where to buy the syringe and how to use it on the leaf so small? thank you !

12/5/2012 11:53:12 AM  

Arsalan Daudi (Author)
Department of Biological Sciences,Royal Holloway University of London

1 ml needleless syringes can be brought from any standard laboratory equipment supplier like Fisher, Sigma etc. These are the very standard syringes with a plunger diameter of about 3 or 4 mm. It doesn't really matter if this plunger diameter varies a bit, as they will usually be much smaller than most Arabidopsis rosette leaves (depending upon your line of course). Please let me know if this answered your question.

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