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Author Topic: Sirius Red Staining  (Read 4688 times)

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Offline SChristina

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Sirius Red Staining
« on: March 05, 2014, 04:47:02 PM »
I have been having problems with my sirius red staining. I have used the following protocol on paraffin embedded liver samples:

1. deparaffinization of slides
2. dehydration
3. picro sirius red for 1 hr
4. 2 changes of acetic acid .5% aqueous solution for 5 min
5. dehydration
6. 2 changes of xylene

The collagen was stained red but so were the nuclei. I want to make it so that none of the nuclei are stained at all. Is there a specific picro-sirius red that is best? Or is there something I can do using the solutions I have now to prevent nuclei staining?

Sirius Red Staining
« on: March 05, 2014, 04:47:02 PM »

Offline gula

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Re: Sirius Red Staining
« Reply #1 on: March 06, 2014, 12:35:45 PM »
I think your stain works well. Basic nucleoproteins, histones, take up the anionic dye. If you want a contrasting colour in the nucleus, you should stain with Weigert's ironhematoxylin (black).
I don't know any method to leave the nuclei unstained, that doesn't also prevent the rest from staining.
gula

Offline immuno

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Re: Sirius Red Staining
« Reply #2 on: March 05, 2015, 11:01:16 AM »
Hi:

My method doesn´t stain nucleous:

Solution 1:0.01% Fast green FCF in saturated picric acid
Solution 2:0,04% Fast green FCF/0,1% Sirius red in saturated picric acid

Stain 15 min in solution 1
without washing stain 15 minutes in solution 2

Dehydrate directly in 100% ethanol (water washes the red stain) , clear and mount

hope this helps

Immuno

Offline NElizabeth

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Re: Sirius Red Staining
« Reply #3 on: April 14, 2015, 09:02:41 AM »
Why are you dehydrating after depar? Is that a typo? Here's our protocol in our path lab where we do hundreds of PSRs.

cut paraffin sections at 5micron
depar: Xylene>Xylene>100%EtOH>100%>95%>95%>70%>H2O
At this point I like to take the slides back to 70% to help them draw in the dye more efficiently. They only need ~2 mins in the 70%.
Then 5-10mins in .1% Sirius Red Picric Acid, we like the one from Rowley Biochemical
Differentiate in 10 dips of 0.1% HCl in H20.
Dip twice in 95% EtOH to clean slides off.
Check under the scope wet to verify your stain. If the smallest fibers aren't staining you can go back in Sirius Red and repeat for another 5mins. (with liver you won't see a lot of staining unless you have a fibrotic model like the CCl4s)
Let slides air dry and then dip once in xylene and coverslip.
You shouldn't be seeing an nuclear staining with the sirius red. 

Re: Sirius Red Staining
« Reply #3 on: April 14, 2015, 09:02:41 AM »