Hi,
I'm going to try an alcian blue/nuclear fast red staining on heart mouse paraffin slides. All the protocols that I've found in internet provide a 30' of incubation in alcian blue. Is this time enough? Is there anybody that can share with me some tricks about this staining protocol? The paraffin section that I'm going to use are very precoious.
Thanks!
You can try the 30 min in alcian blue. Then rinse in water and look at the slide in the mikroskope. If the stain is not intense enough in your opinion, put it back again in the staining solution.
Check the pH of the staining solution. It should be 2,5 for the "normal" alcianblue. If it has pH 1, it stains only sultated proteoglycanes --> less colour in your slide.
If you have the possibility use a positive control like salivary gland or small intestine. This gives really strong alicianblue. Alcianblue solutions are used in the range from 0,1-1%. We incubate our slides for 10 min in 1% alcianblue.
Be sure to use the Nuclear fast red - aluminium-sulfate - lake for counterstaining. - not the nuclear fast red alone. Nuclear fast red is an acid dye, that acts like eosin. It has to be combined with a mordant like aluminium-sulfate. Then the overall ionisation is positive and it stains nuclei.
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/n8002pis.pdf
If you are not happy with the ready stain, put the slides for a short time in acid-ethanol to destain them again. Rinse in water and begin from the start.
Hi,
we used this stain in the past above all for staining alginate-embedded chondrocytes derived from stem cells (cardiac and umbilical cord). You may find the protocol and images in this paper available from my profile
https://www.researchgate.net/publication/231212904
The timing we used was 30'
Good luck
Giampiero
Article Isolation and Characterization of CD276+/HLA-E+ Human Subend...
30' Alcian blue is enough. Only problem with heart sections is that the nuclear fast red is not very nuclear, but also largely labels all cardiomyocytes. We observed this problem only in heart. If you have very precious samples I would re-think strategy.
You can try the 30 min in alcian blue. Then rinse in water and look at the slide in the mikroskope. If the stain is not intense enough in your opinion, put it back again in the staining solution.
Check the pH of the staining solution. It should be 2,5 for the "normal" alcianblue. If it has pH 1, it stains only sultated proteoglycanes --> less colour in your slide.
If you have the possibility use a positive control like salivary gland or small intestine. This gives really strong alicianblue. Alcianblue solutions are used in the range from 0,1-1%. We incubate our slides for 10 min in 1% alcianblue.
Be sure to use the Nuclear fast red - aluminium-sulfate - lake for counterstaining. - not the nuclear fast red alone. Nuclear fast red is an acid dye, that acts like eosin. It has to be combined with a mordant like aluminium-sulfate. Then the overall ionisation is positive and it stains nuclei.
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/n8002pis.pdf
If you are not happy with the ready stain, put the slides for a short time in acid-ethanol to destain them again. Rinse in water and begin from the start.
If your samples are really important, checking them every once in a while as mentioned above is probably a good idea, so you don't overstain them.
Best of luck.
1-Take sections to water
2-Stain in alcian blue solution pH 2.5 for 5 minutes
3-Wash well in water
4-Counterstain in 0.1% nuclear fast red for 5 minutes
5-Wash in water
6-Dehydrate, clear . Mount sections in DPX
Mucins - Bright Blue
Nuclei - Red
To further identify specific types of acid mucins you need to use the other pH solutions.
Strongly sulphated mucins - use pH 0.2 Alcian Blue
Weakly sulphated mucins - use pH 2.5 or pH 1.0 Alcian Blue
Best of luck
pH is very critical when doing Alcian Blues . Follow the method of El -Sayed , but instead of washing with water after Alcian Blue staining. Rinse with the diluent that the Alcian Blue was dissolved in. For example Alcian blue with a pH of 2.5 is made in a 3% Acetic acid. Then wash the slides in 3% Acetic acid after you have stained them with Alcian Blue. Counterstain with nuclear fast red.
Just as an information to interested colleagues:
I guess, you all are talking about the 'modified Alcian Blue technique' (MOWRY 1956).
cf: BANCROFT JD & GAMBLE M.
(Eds, Theory and practice of Histological Techniques, 8th ed, print 2011) p. 172-173.
That recipe uses
1% Alcian Blue (in 3% Acetic acid solution, = pH 2.5) staining for 30 minutes, rinsing afterwards in running tap water for 5 minutes. The hint for using the NFR-lake (combined with Al-sulfate [ 5g Aluminium sulfate Al2(SO4)3.18H2O to be dissolved in water with heat; add the NFR to water while still hot and filter) , 100 ml deionized water, 0.1g Nuclear Fast Red] is imperative. Counterstaining in this NFR-lake for 10 minutes, washing in running tap water for 1 minute. Dehydrate in graded ethanols ....xylene, mount in a miscible medium.
I second Gudruns valuable informations on performing in first place and (if available) also in parallel controls (and / or the staining method first to be tried on sections not so valuable for your study...).
Good luck and best wishes.
I recommend that you follow the answer of Gudrun Lang.
Good luck
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