I already tested aniline blue and fast green instead of Astra blue but the results were not satisfactory.
Distinguishing between lignified and non-lignified cells is the main reason of this double staining; especially between cambium cells and mature xylem.
If a temporary staining is acceptable for you (e.g. when you take digital images of your samples) you may use phloroglucinol-HCl. Lignified tissue is stained reddish, all other tissue remains unstained.
Safranine-Astra blue combination is to distinguish lignified (stained by safranine) and non-lignified (Stained by Astra blue). The best alternative is Safranine O-Fast green FCF where fast green should be prepared in a mixture of ethanol:methyl cellosolve:clove oil (1:1:1). You should get satisfactory results if these mordant have mixed while preparing fast green. Staining duration is similar to that of Safranine-Astra blue.
If you want to get a clear image of the cell wall of cambial cells and xylem cells distinctly, you can also use Tannic acid:Ferric chloride-Lacmoid staining. This will not stain the cytoplasm and will give more contrast to cell wall. Primary wall will appear brownish while lignified secondary wall will appear Blue. This is the best staining method for cambial cell wall. But it takes 3-4 days of staining in lacmoid for the best results.
I I really appreciate both above answers; they were really helpful. I had heard about "Wiesner reaction" and similar staining (Mäule reaction) but as you mentioned preparing permanent slides is impossible by using these methods. I didn't know about preparation method of Fast green; I simply used the water soluble FCF! I will definitely try the suggested methods. Thank you both again.
Hi Reza
As i know, Safranin and fast green could be best staining for your anatomy study, it takes a bit of time but you will get a good result.
good luck with my regards
Abdul
Dear Reza
I know the procedure for that, i am happy to send it to you if you like.Regards
Abdul
Dear Badi,
That would be so nice of you if you send me the staining protocol. my e-mail address: [email protected]
Dear Raza,
Safranin - fast green is one of the best combination for woody tissue.
Toludine blue at ph 4 will get a best result for woody tissue in various grade of blue colour to lignified and non lignified tissues
Safranin - fast green will give you the best result if astra blue fails in the double staining of woody tissue. try.
Hi Reza,
Here the protocol I use with a mix of Safranin and Alcian blue, works well.
Success!
Dear Nele,
Thank you for sharing the file. That is also a nice teaching source. However, I should search to see if Alcian blue is available around.
regards
Dear Nele,
despite NOT working with wood or plants (human tissue for diagnostic ultrastructural evaluation) I would like to express my sincerest thanks for this really impressive collection of your wood sampling, processing and staining protocol. This is what (Histo-Ultrastr.) methodologists (which I am too) like. Congratulations and THANK you again,
Wolfgang MUSS, Salzburg
AUSTRIA
We use cresyl-violet (0,05 % water solution), what permits to receive different colorations with the cells at different stage development. We checked this stain during study of wood formation in conifers and oak.
We use cresyl-violet (0,05 % water solution), what permits to receive different colorations with the cells at different stage development. We checked this stain during study of wood formation in conifers and oak.
For paraffin embeded sections of many organs, I have been obtaining great results with the metachromatic toluidine blue O.
1) Stain your sections before removal of the paraffin for 30 minutes.
2) Rinse slides with tap water and air dry. Dehydration with alcool will remove the color!
3) Remove paraffin with xylene and mount couverslip.
Lignified walls will be blue and non-lignified, purple.
Did it ever happen to any of you that after clearing the slides with Xylol, the staining turned badly dark?? or any idea why this could happen??
@Camila
If excess stain is not removed during rinsing with water and ethanol, after immersing in xylol, the liquid will be obscure or dark. In this case, I rinse or soak sections in fresh xylol for one or two more times till it gets bright. I should add that sometimes I get dark sections and figured out that the problem is used adhesives. Anyway, using xylol is not always obligatory and if sections stay long enough in ethanol 100%, the final quality will be as good.
Before pure xylene trasfer the slide to carbol xylol for 2 min then to xylene . The time of detaining of excess stain in 30% tp 70% alchohol just increase. if it is 1 min try 2-3min and check whether the stain removed from unwanted areas. After double staining using alcohol/ clove oil stain you place 2 step of destaining solution of either 100% alcohol or clove oil then change to xylene series. This is found to be a good method for me.
Hello
I already wrote that we used aqueous solution 0.05% cresyl violet acetate. In this case it is well soluble in water and gives clear image of cell walls of cambial zone and other developmental zones of xylem and phloem. Different staining of cell walls on microcore sections and chemical reasons of that we described in our paper, published in 1981year (n 4, p.102-105) in journal "Chemistry of Wood". Although the article was published in Russian I attach her now
Best Regards,
Galina
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