Dear Dr.,
For staining plant cell nuclei in leaf and stem samples prepared with a microtome, a commonly used and simple chemical reagent is acetocarmine. This stain is widely utilized in plant cytology due to its specificity and ease of use.
Why acetocarmine is suitable:
Acetocarmine Staining
Properties:
- Specificity: Acetocarmine specifically stains chromatin and other nuclear materials, making the nuclei clearly visible under a microscope.
- Contrast: It provides a good contrast against the cytoplasm and cell walls, enhancing the visibility of the nuclei.
- Ease of Use: The staining procedure with acetocarmine is straightforward and does not require complex preparation or handling.
Staining Procedure
1. Preparation of Acetocarmine Solution:
- Dissolve carmine in glacial acetic acid to prepare the acetocarmine solution. A typical concentration is 1-2% carmine.
2. Staining Process:
- Place the plant tissue sections on a microscope slide.
- Add a few drops of the acetocarmine solution onto the sections.
- Allow the stain to penetrate for a few minutes to ensure thorough staining of the nuclei.
- Rinse gently with distilled water to remove excess stain, if necessary.
3. Observation:
- Mount the stained sections with a coverslip and observe under a microscope.
Alternatives
While acetocarmine is a popular choice, other stains can also be used for staining plant cell nuclei:
- Hematoxylin: Commonly used in combination with counterstains like eosin.
- DAPI (4',6-diamidino-2-phenylindole): A fluorescent stain that binds strongly to DNA, useful for fluorescence microscopy.
Each of these stains has its own advantages and specific applications, but for simplicity and routine use, acetocarmine remains a preferred option.
Good luck.
Safranin (or safranin O) is a nuclear dye. It colours cell nuclei red, and is mainly used as a counterstain.
Hi Faride,
Do you want to image with normal brightfield microscopy or with fluorescence microscopy? And Do you want to prepare permanent sections (dehydrated and mounted in a resin such as Entellan or very old-school canada balsam) or is that not important to you? Also do you want to stain any other structures? Depending on these answers, the staining methods might change.
In principle, there are many dyes that can stain nuclei selectively. Some of the easiest for fluorescence microscopy would be DAPI or Hoechst dyes. Methyl green also works well as a fluorescent nuclei stain with red excitation and far-red emission.
For brightfield, you can use either method already mentioned in the other comments. Hematoxylin or iron hematoxylin would probably work as well, although I have no experience with plant material in particular... I would assume for a nuclei staining that wouldn't matter. Those stainings are highly robust and easy to do.
Depending on other stainings you might want to do on the same sections, the method might also change. For brightfield microscopy, you obviously need to select a nuclear dye of a different colour than the other staining. Azocarmin is red, hematoxylin is blue and iron hematoxylin is dark brown to black. Methyl green at higher concentrations can also be used as a nuclear staining in green, but can be tricky to process without washing it out and making it too faint.
That should give you enough flexibility for any other staining you'd wish to do.
Hope this helps!
Cheers,
Sven