[Histonet] PicroSirius Red in Frozen Sections

[Lee & Peggy Wenk]

I'm going to try to take a stab at this, but I don't know specifically about 
the picrosirius red stain. I'm going to talk in general about stains and 
fixation (or lack thereof in the frozen section (FS)), and the relationship 
between fixation and staining. I'm going to start out very simple, and then 
get a little more complicated, so hang in there. (If you are not interested 
in dye theory, delete now, as this is going to get long. But if you like dye 
theory (like I do), I hope you enjoy this. And if you do like dye theory, 
let me know if you think I'm correct, or if I’m barking up the wrong tree.)

Stains bind to proteins in the tissue, which are made up of amino acids, 
which either are positively charged, negatively charged, or non-polar (no 
charge). Dyes usually have positive ions and negative ions, but tend to have 
more of one type, and thus are considered either positively charged or 
negatively charged, and/or tend to bind via one type of bonding over another 
(e.g., hydrogen bonds vs. covalent).

Different Proteins in tissue have their own unique shape and density. Dyes 
have their own shapes. The two shapes have to be somewhat similar for the 
proteins to bind to a dye. A round ball might be able to fit into an open 
space in a protein, whereas a stiff long dye (think something the shape of a 
ruler) might not be able to fit into that space.

Differently Proteins have their own density (loose to tightly packed). Dyes 
have their own sizes (small to very large). The dye has to be able to fit 
into the protein, so a large dye may find it difficult to fit into a dense 
bunch of protein, whereas a small dye can fit into loose protein and dense 
protein.

Therefore, for a dye to bind to a protein, the charged ions on the dyes have 
to be the opposite of the charges on the amino acids/proteins (positive 
binding to negative). And the charges on the dyes have to line up with the 
charges on the proteins, so the dye has to be able to "fit" into the 
protein, and the charges have to line up.

Therefore, if we do something that changes the CHARGES on the protein, 
and/or the SHAPE of the proteins, and/or the DENSITY of the proteins, the 
dye may bind differently (not at all, very little, or too much). 
(Conversely, changing the dye in any way could cause different staining 
patterns, but since you said it was the same kit, and since you said "later" 
your boss asked for the stain to be done on a FS, I'm expecting it to have 
been the same day or the next, so I don't think the kit went bad, and I'm 
assuming you did the stain correctly. So I won't be discussing bad staining 
due to bad dyes or performing the stain incorrectly.)

Now, onto fixation vs. frozen section (FS). I'm assuming 10% formalin was 
the fixative, or a zinc formalin, or a formalin substitute (glyoxal). It's 
the formalin/formaldehyde/glyoxal that is negatively charged. It will bind 
with positive amino acids in the protein. Let's assume there were 10 + and 
10 - amino acids on the protein, so the overall charge of the protein is a 
net zero. Let's bind 4 of the + amino acids with - charged formalin. You now 
have 6 + and 10 - amino acids, so you have more negative amino acids than 
positive, so your tissue is more negatively charged. You have just changed 
the CHARGES on the proteins.

Fixatives cross-link proteins, and pull them in different directions. You 
have therefore changed the SHAPE of the protein.

Since the fixative is cross-linking the protein, and pulling the proteins in 
different directions, some proteins are going to be pulled further apart, 
thus becoming looser in density, while other proteins are being pulled 
closer together, or being made denser. You have therefore changed the 
DENSITY of the protein.

Most dyes/stains used in histology were designed to be used with formalin 
fixed tissue, and are therefore made to work with proteins that have had 
their charges changed, their shapes altered, and their density changed, 
according to the changes made by formalin.

Frozen sections have NOT have any fixative, and are therefore similar to the 
unfixed tissue, without the changes in charged, shape, and density. So it 
should make sense that unfixed/FS tissues should/could stain differently 
than fixed tissue.

Now, for sirius red specifically. There are several different sirius red dye 
molecules. I don't know which one in particular you used, but in this 
explanation, it doesn't really matter, because they all belong to the 
polyazo dye family. That means they are made up of several benzene rings (5 
to 8), held together in a long row (linear - like the ruler I mentioned 
earlier) with azo bonds (Nitrogen double bonded to Nitrogen -N=N- which have 
hydrogens bonded to them, giving these bonds a positive charge), and several 
sulfonic acid groups bound to the benzene rings ( -SO3 ions, which are 
negatively charged).

To me, these sirius red dye molecules look very similar to Congo red dye 
molecules, and according to Conn's Biological Stains, sirius red binds in a 
similar manner to Congo red.

Therefore, these long linear dye molecules have to bind via hydrogen bonds 
(positive charges) to very specifically spaced negative charges (usually 
hydroxyl ions -OH) on the connective tissue. When these dyes bind, they do 
so in a way that the dyes are now parallel to another  | | | | | | .  This 
makes a crystal-like arrangement, and these dyes/connective tissue (with 
sirius red) or dyes/amyloid (with Congo red) will birefringe with polarizing 
microscopes.

These specifically spaced hydrogen bonds between the dye and the protein 
will line up on FORMALIN FIXED tissue, where the connective tissue (or 
amyloid) have been cross-linked to make a specific shape, specific density, 
and the correct number of hydroxyl ions are in the correct position.

However, in the case of FS, the protein shape, density, and number and 
position of hydroxyl ions are different than on formalin fixed tissue. In 
the case of your sirius red, since there are more negative amino acids on FS 
than on formalin fixed (in relation to the number of available positively 
charged amino acids), it might explained why the tissue looks redder (more 
hydrogen bonding of sirius red to negatively charged amino acids). However, 
the shape of the protein on FS is different than on the formalin fixed 
tissue, so the dye may not be binding | | | | |, but may be binding | \ / _ 
_ _ | /_. So, even though there are more sirius red dye molecules binding, 
they are not doing so in a parallel crystalline fashion. Therefore, there 
will not be polarization of the dye molecule.

Does this long-winded explanation make sense?

Now, if the problem is just overstaining, then more differentiation would 
help. But if the problem is the shape of the connective tissue proteins and 
the spacing of the hydroxyl ions, then nothing will help.

However, like I said, I don't really know this stain, so I don't know if it 
really SHOULD work on FS or not. I'm just trying to explain why it may not 
be working. I know that with Congo red, for us, the staining actually seems 
better on FS than on formalin fixed, paraffin embedded tissue. But we do get 
more connective tissue picking up Congo red on FS. So that can be a problem 
for us.

Peggy A. Wenk, HTL(ASCP)SLS

-----Original Message----- 
From: Yoanna Bello Arredondo
Sent: Monday, November 11, 2013 9:37 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PicroSirius Red in Frozen Sections

Hello!

I did a Picrosirius red stain using the polysciences kit in paraffin section 
cut at 4microns. The stain looks great. Later on my boss asked me to do the 
picro using the same kit but in frozen section. The stain looks very, very 
intense. Sections are really red. When looking under the brightfield 
microscope there is not yellow, the entire tissue is red. Under the 
polarized microscope, we can see the fiber but no  like the paraffin 
sections. Does anybody have come across this situation before? Does the 
picrosirius stain only works for paraffin sections? I will appreciate any 
feedback on the matter. I have been searching online for a protocol of 
Picrosirius stain for frozens, but no luck.

Thank you in advance,



Yoanna Bello

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