[Histonet] Fast Green / Sirius Red - Unknown blue features

Thu Jun 22 09:36:57 CDT 2023

David

We have found that mordanting in bouins (just like you would a Masson's trichrome) prior to completing a standard Picrosirius Red stain protocol that we get very consistent results.  I think this method has been published but I do not have access to the reference currently.  Its worth a shot for your PSR stain.  What I do know is that the brief rinse in acetic acid post the PSR stain is necessary, that seems to remove any excess red/pink that may stain the cytoplasm of the cells, not much of a distractor for manual reads for fibrosis but may be problematic if you are utilizing an image analysis solution.  I can't comment on the other method.  If you like I can forward on our PSR method in a separate email.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz at premierlab.com
www.premierlab.com

Take a look at our most recent publications:

https://journals.lww.com/appliedimmunohist/Abstract/publishahead/An_Image_Analysis_Solution_For_Quantification_and.98711.aspx

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879147/

-----Original Message-----
From: David Burk via Histonet <histonet at lists.utsouthwestern.edu> 
Sent: Thursday, June 22, 2023 6:38 AM
To: John Kiernan <jkiernan at uwo.ca>; histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] Fast Green / Sirius Red - Unknown blue features

Dr. Kiernan,

You are correct regarding the source of the technique we are emulating. We have had issues with consistency in keeping the yellow picric acid color in cytoplasm when performing the traditional PSR stain possibly due to variability in what each individual considers 'quick' in their rinses and ethanol steps. Looking in the literature, you can find many publications that show PRS-stained tissue with golden-yellow / straw-colored cytoplasm (ideal), pale pink, or even virtually 'clear'.

The stain we are currently using is from a bottle likely purchased in the 80's or 90's and does bear the label you describe. Allied Chemical in Morristown, NJ which no longer exists. I'll try a newer bottle we have and see if the results remain the same. Our Direct Red 80 is from Sigma-Aldrich (365548) and does not bear the certification mark you mention. However, as we do see the expected collagen staining in tissues (and the stained material exhibits birefringence), I am fairly confident that it is, at the very least, 'OK'. If you happen to know of vendors that routinely offer certified dyes, I'd be happy to utilize them in the future.

As you mention in your separate email, what I may consider blue may appear differently to others either by technology or anatomy/physiology. Regardless, they are unexpected and have not been described in the literature and, in that regard, present a potential source of confusion.

Best,
David

________________________________
From: John Kiernan <jkiernan at uwo.ca>
Sent: Wednesday, June 21, 2023 11:36 PM
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>; David Burk <David.Burk at pbrc.edu>
Subject: Re: Fast Green / Sirius Red - Unknown blue features

Your technique is the one first (I think) published by Lopez-De Leon A & Rojkind M (1985) A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J. Histochem. Cytochem. 33: 737-743. The photos in that paper show some of the collagen almost black - surely taking up both red and green dyes. More recent papers describe exactly the same method, and there are also some variants. Your technique, with an acid rinse after staining for an hour, then quick transition to rapid dehydration in 100% alcohol, is essential for any valid picro-sirius staining.

According to the entry for fast green FCF (CI 42053) in Conn's Biological Stains (10th ed, p.180-182), "chemically distinct blue-green dyes have been supplied under this name". Are you sure your fast green FCF is the real McCoy? Is it from a batch certified by the Biological Stain Commission? The jar of dye powder should have a small bluish label, with features that make forgery difficult. See https://us-west-2.protection.sophos.com?d=biologicalstaincommission.org&u=aHR0cHM6Ly9iaW9sb2dpY2Fsc3RhaW5jb21taXNzaW9uLm9yZy9jZXJ0aWZpZWQtYmlvbG9naWNhbC1keWVzc3RhaW5zLw==&i=NWYzYTk2OTEyMDVmMDkwZWJiNmJlNzc2&t=WjVWeGtKVHl5R3FneU9tQjkxK1gydnlWY2Y4Yk9ESGZ1SjBkY1c3ZmxRST0=&h=279d4c37c7764be8a8cd92a53a018013&s=AVNPUEhUT0NFTkNSWVBUSVZpNWvhfkvsy0HBXeMJqMJXLVUzTeOGssiQ7jAnNTZ6aDtn6HKp77NuWaELZg1b57k for pictures and other information. There are companies selling "certified stains" that have not been tested and certified by the Biological Stain Commission. Caveat emptor!

The Biological Stain Commission is a not-for-profit corporation that has been providing third-party quality control and other services for vendors and users of stains for 100 years.

Just a few thoughts; I could add more, but probably this letter already is too long for the Histonet censors.

John Kiernan
Professor Emeritus, Anatomy & Cell Biology University of Western Ontario, London, Canada https://us-west-2.protection.sophos.com?d=uwo.ca&u=aHR0cHM6Ly93d3cuc2NodWxpY2gudXdvLmNhL2FuYXRvbXkvcGVvcGxlL2Jpb3MvZW1lcml0aS9raWVybmFuX2pvaG4uaHRtbA==&i=NWYzYTk2OTEyMDVmMDkwZWJiNmJlNzc2&t=NjhVVUJSWDJ5TlVOTHJ0QmgyTlNjd0dzK0ZUV1kyVEFHMnJlMmVLbS82ST0=&h=279d4c37c7764be8a8cd92a53a018013&s=AVNPUEhUT0NFTkNSWVBUSVZpNWvhfkvsy0HBXeMJqMJXLVUzTeOGssiQ7jAnNTZ6aDtn6HKp77NuWaELZg1b57k
Also  Secretary, Biological Stain Commission, Inc.
https://us-west-2.protection.sophos.com?d=biologicalstaincommission.org&u=aHR0cHM6Ly9iaW9sb2dpY2Fsc3RhaW5jb21taXNzaW9uLm9yZw==&i=NWYzYTk2OTEyMDVmMDkwZWJiNmJlNzc2&t=VlpjOEZ2cXZPQ3hCeGxVMTJqbWExbnlyemQ1alREREhnK3dYMk5WMy80dz0=&h=279d4c37c7764be8a8cd92a53a018013&s=AVNPUEhUT0NFTkNSWVBUSVZpNWvhfkvsy0HBXeMJqMJXLVUzTeOGssiQ7jAnNTZ6aDtn6HKp77NuWaELZg1b57k
= = =
________________________________
From: David Burk via Histonet <histonet at lists.utsouthwestern.edu>
Sent: June 21, 2023 5:48 PM
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Fast Green / Sirius Red - Unknown blue features

We are working out an alternative method for collagen staining using Fast Green / Sirius Red (Direct Red 80) rather than the standard picrosirius red method as I think it is prettier and easier to see the collagen on a green background.

What we've noticed, though, is that we are seeing intermediate blue staining in the tissue in particular regions or structures. I've not had any success in finding an explanation for this online and was hoping someone on the board may have an idea of what's going on and what is being stained blue in our tissue sections.

We have examined a variety of murine or rat tissues including liver, heart, kidney, lung, skeletal muscle, spleen, brain, pancreas, and even decellularized human adipose tissue. There are, almost always, some structures/features that exhibit a denim blue to lighter blue-green color (at least to my eye) in addition to the expected red-colored structures that we would assume to be collagen, light green cytoplasm, and yellow-ish features stained with picric acid.

An interesting tidbit is that these blue-ish stained features are birefringent under polarized light so you would not know their color (with transmitted imaging) was atypical.

I don't want to use a stain if I can't let people know what a particular color represents and can also cause problems with the quantification of collagen using a color-based approach.

Our protocol is as follows:

  1.  Dewax
  2.  H2O rinse
  3.  Stain in a 0.1% Fast Green FCF (C.I. 42053) and 0.1% Direct Red 80 (C.I. 35780) solution dissolved in saturated picric acid for 1 hour at room temperature
  4.  Dip 5x and then immerse in 0.5% acetic acid for 5 seconds
  5.  Repeat step 4
  6.  Dip 5x and then immerse in 100% Ethanol 30 seconds
  7.  Dehydrate in 100% Ethanol 1 min
  8.  Repeat step 7
  9.  3 x Xylene for 2 min each
  10. Coverslip

I'm uploading some images from mouse muscle and tumor tissue to the Histonet Image upload site. If that doesn't work, here are links:

Mouse tumor:

https://us-west-2.protection.sophos.com?d=google.com&u=aHR0cHM6Ly9kcml2ZS5nb29nbGUuY29tL2ZpbGUvZC8xVmpPWkZ6dnNRQnlRTHVEdGRHUGZkd0F3YXBfQ1ZFNTgvdmlldz91c3A9c2hhcmluZw==&i=NWYzYTk2OTEyMDVmMDkwZWJiNmJlNzc2&t=dlN6YnhXQXI0UHBZSVRCb2JxY1hLU054WHZwUGtoc2xXZWN4eUpoUXN4cz0=&h=279d4c37c7764be8a8cd92a53a018013&s=AVNPUEhUT0NFTkNSWVBUSVZpNWvhfkvsy0HBXeMJqMJXLVUzTeOGssiQ7jAnNTZ6aDtn6HKp77NuWaELZg1b57k

Mouse skeletal muscle:

https://us-west-2.protection.sophos.com?d=google.com&u=aHR0cHM6Ly9kcml2ZS5nb29nbGUuY29tL2ZpbGUvZC8xMHZUX0ZLdTMtQWQ1dWVtTTVnblptcURDS2IzZnMybFYvdmlldz91c3A9c2hhcmluZw==&i=NWYzYTk2OTEyMDVmMDkwZWJiNmJlNzc2&t=OXpFOVZvNzV4cDYvN2R6bkhiNFh3SFAyaHBTc3NsTDJYOFFaZTBTRDI3OD0=&h=279d4c37c7764be8a8cd92a53a018013&s=AVNPUEhUT0NFTkNSWVBUSVZpNWvhfkvsy0HBXeMJqMJXLVUzTeOGssiQ7jAnNTZ6aDtn6HKp77NuWaELZg1b57k

Thanks,

David Burk

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