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

[David Burk]

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://drive.google.com/file/d/1VjOZFzvsQByQLuDtdGPfdwAwap_CVE58/view?usp=sharing

Mouse skeletal muscle:

https://drive.google.com/file/d/10vT_FKu3-Ad5uemM5gnZmqDCKb3fs2lV/view?usp=sharing


Thanks,

David Burk