Mar 09, 2026
Safranin-O/Fast Green Staining of Clinical Tissues in Formalin Fixed Paraffin Embedded (FFPE) Samples
- Ashleigh Abbott1,
- Robert Dalton1,
- Lauryn Lafayette1,
- Shane Priester1,
- Folly Patterson1,2,
- Carlos Cruz3,2,
- Kiara Chan1,
- Kyle D. Allen1,3,4
- 1UF Department of Biomedical Engineering, College of Engineering;
- 2Department of Community Dentistry & Behavioral Science, College of Dentistry;
- 3UF Pain Research and Intervention Center of Excellence;
- 4Orthopedic and Sports Medicine Institute, College of Medicine
Protocol Citation: Ashleigh Abbott, Robert Dalton, Lauryn Lafayette, Shane Priester, Folly Patterson, Carlos Cruz, Kiara Chan, Kyle D. Allen 2026. Safranin-O/Fast Green Staining of Clinical Tissues in Formalin Fixed Paraffin Embedded (FFPE) Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9n354l3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 06, 2026
Last Modified: March 09, 2026
Protocol Integer ID: 269394
Keywords: Tissue staining, Knee Joint, Formalin fixed, Paraffin embedded, Safranin O, Fast Green, Cartilage, Osteoarthritis, Formalin Fixed Paraffin Embedded, FFPE, Imaging, knee replacement, imaging cartilage, cartilage degradation, OARSI, clinical samples, post-fixed clinical tissues, fast green staining of clinical tissue, osteoarthritis, cartilage interface in knee joint, staining clinical ffpe sample, stage of osteoarthritis, knee joint, cartilage component, fast green staining, using histological stain, grading bone, additional representation of proteoglycan proportion, bone content, cartilage interface, histological stain, proteoglycan content, staining, stain, clinical tissues in formalin fixed paraffin embedded, proteoglycan proportion, bone for an oarsi score, clinical ffpe sample, bone, safranin, clinical tissue
Funders Acknowledgements:
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Grant ID: UC2AR082196
Abstract
In troubleshooting examples, this stain is utilized when grading bone for an OARSI score. Saf-O/Fast Green helps not only in determining stage of osteoarthritis, but also for looking at the proteoglycan content (stained with safranin). This protocol describes the stepwise process for staining clinical FFPE samples to visualize cartilaginous and bone content using histological stains, Saf-O and Fast Green respectively. This method clearly visualizes the bone-cartilage interface in knee joints, with additional representation of proteoglycan proportion in cartilage components, indicated by the intensity of red hues.
Materials
- Slide holder cassettes
- Histology staining bins
- Slide coverslips
- Eyedropper
- Mounting medium (Permount)
- Fume hood
- Protocol™ SafeClear Xylene Substitute (Fisher Healthcare, 68551-16-6, 11-002-220)
- Permount Mounting Medium (Fisher Chemical, 108-88-3, 68240-09-5, SP15-500)
- 1050mL Ethanol
- 550mL Distilled Water
- 500mL Xylene substitute
- 50mL Hematoxylin A
- 50mL Hematoxylin B
- 0.1g fast green powder
- 1.00g safranin-o powder
- 1mL Glacial acetic acid
- 1mL Hydrochloric acid
Troubleshooting
Safety warnings
NOTE: The intensity of red staining safranin-O varies depending on how long the samples are stained for. When troubleshooting the protocol, try increasing the time of the safranin-O staining, lying the slides horizontally and using an eyedropper to add the stain (may work better than the vertical slide holders), and/or lying the slides on the slide warmer (heat will increase staining intensity).
NOTE: if staining is not going well (and you are following the protocol accurately) – it is likely a processing (decal/infiltration/fixation) issue. Don’t make permanent changes to the protocol if this is the case.
NOTE: Solution prep is based on solution containers that fully submerge all sections on all slides being stained when filled with ~100mL. Ensure containers meet these standards or scale solution ratios to match these requirements.
Ethics statement
Tissue collection for this protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Before start
NOTE: solution prep is based on solution containers that fully submerge all sections on all slides being stained when filled with ~100mL. Ensure containers meet these standards or scale solution ratios to match these requirements.
Reagents
| Name | Vendor | CAS number | Catalog Number or Product Code | RRID | |
| Fast Green Powder | Thermo Scientific | 2353-45-9 | A16520.14 | ||
| Fast Green Powder | Thermo Scientific | 477-73-6 | 146641000 | ||
| Fast Green Powder | Electron Microscopy Sciences | 64-17-5 67-56-1 517-28-2 | 26370-03 | ||
| Fast Green Powder | Electron Microscopy Sciences | 7647-01-0 | 26458-05 | ||
| Fast Green Powder | Decon Laboratories | 64-17-5 | UN1170 | ||
| Fast Green Powder | Alfa Aesar | 64-19-7 | UN2789 | ||
| Fast Green Powder | Fisher Chemical | A144S-500 | 7647-01-0 | ||
| Fast Green Powder | Fisher Healthcare | 68551-16-6 | 11-002-220 | ||
| Fast Green Powder | Fisher Chemical | 108-88-3 68240-09-5 | SP15-500 |
Troubleshooting
Hematoxylin: nuclei stain, positively charged dye that binds to negatively charged phosphate groups of the nucleic acid of the cell nucleus, staining the nuclei dark blue to dark violet.
Safranin-O: positively charge, cationic dye (basic dye) that stains acidic (negatively charged) proteoglycans and glycosaminoglycans present in growth plate cartilage and articular cartilage varying shades of red.
Fast Green: counterstains non-collagen sites and provides a contrast to Safranin-O.
Above is an example of a properly stained section.
The intensity of red staining safranin-O varies depending on how long the samples are stained for. When troubleshooting the protocol, try increasing the time of the safranin-O staining, lying the slides horizontally and using an eyedropper to add the stain (may work better than the vertical slide holders), and/or lying the slides on the slide warmer (heat will increase staining intensity).
If staining is not going well (and you are following the protocol accurately) – it is likely a processing (decal/infiltration/fixation) issue. Don’t make permanent changes to the protocol if this is the case.
Tips
Use the same Fast Green and Saf-O for your entire study.
Make enough that you will not run out.
Make it fresh before starting (do not use old solutions if this is for grading/papers/etc.).
Fast green and Safranin-O solutions can be stored and used for longer than a week.
Wrinkles in cartilage, try drying slides between steps.
Solution Preparation
1h
Set aside from stock 400mL of 100% Ethanol
- Separate into 100mL aliquots
Using distilled water, mix from stock 400mL of 95% Ethanol (19:1)
- Separate into 100mL aliquots
Using distilled water, mix from stock 100mL of 80% Ethanol (4:1)
Using distilled water, mix from stock 100mL of 50% Ethanol (1:1)
Combine 50 mL of Hematoxylin A and 50 mL of Hematoxylin B for 100mL of Weigert’s Hematoxylin (1:1)
Wrap bottle in foil to prevent bleaching of dye after mixing
- Solution can be used for first week after mixing, after which it becomes ineffective
On a stir plate with a magnetic stir bar, add 0.10g of fast green powder into a beaker containing 100mL of distilled water (1mg/mL)
- Allow powder to dissolve 10 minutes for a 0.10% Fast Green Solution
On a stir plate with a magnetic stir bar, add 1.00g of safranin-o powder into a beaker containing 100mL of distilled water (10mg/mL)
- Allow powder to dissolve 10 minutes for a 1% Safranin-O Solution
Mix 70mL of 100% Ethanol, 30mL of distilled water, and 1mL of Glacial acetic acid for a ~1% Acetic Acid solution (7:3:1)
Mix 70mL of 100% Ethanol, 30mL of distilled water, and 1mL of Hydrochloric Acid for a ~1% Acid Alcohol solution (7:3:1)
Also Prepare:
- Five 100mL aliquots of Xylene substitute
- Two 100mL aliquots of Distilled water
Set-up
30m
Have your chosen slides picked out and loaded into their slide holder
Set up your work station in the fume hood with solution containers lined up in order for the processing steps:
- Xylene substitute (3)
- 100% ethanol (2)
- 95% ethanol (2)
- 80% ethanol (1)
- 50% ethanol (1)
- Distilled water (1)
- Weigert’s hematoxylin (1)
- Acid Alcohol (1)
- Distilled water (1)
- Fast Green (1)
- Acetic Acid (1)
- Safranin-O (1)
- 95% ethanol (2)
- 100% ethanol (2)
- Xylene substitute (2)
Label and fill solution containers with 100mL of their appropriate solutions
Deparaffinize and hydrate slides
39m
To remove paraffin, soak slides in Xylene for 18 minutes, switching containers twice so 3 batches of Xylene each saturate the slides for 6 minutes each.
18m
Once the slides have sat in the first Xylene container for 6 minutes, remove and place them in the next container for 6 minutes
Repeat once more.
Next steps will gradually hydrate the slides.
Leave slides in 100% ethanol for 6 minutes, switching containers 3 minutes after the slide holder is placed in the first batch.
6m
Leave slides in 95% ethanol for 6 minutes, switching containers 3 minutes after the slide holder is placed in the first batch.
6m
Place slides in the next container of 80% ethanol for 3 minutes.
3m
Now, move slides to the container of 50% ethanol for another 3 minutes.
3m
Complete the hydration steps, moving the slide holder to Distilled water for a final 3-minute interval.
3m
Staining tissue steps
21m 25s
Stain slides in Weigert’s hematoxylin for 10 minutes.
10m
Dip slides in 1% Acid Alcohol once.
Rinse slides in water for 30 seconds.
30s
Stain slides in 0.10% Fast Green for 45 seconds.
45s
Rinse in 1% Acetic Acid for 10 seconds.
10s
Stain slides in 1% Safranin-O for 10 minutes.
10m
Dehydrate tissues
2m
To dehydrate slides, dip slides in 95% ethanol 5-10 times.
30s
Repeat in final 95% ethanol container
30s
Now, dip slides 5-10 times in 100% ethanol.
30s
Repeat in final 100% ethanol container.
30s
Clear tissues
1m
Clear slides, dipping 5-10 times in Xylene substitute container
30s
Container should appear clouded with stain after being dipped.
In final, clean, Xylene containers repeat 5-10 dips of slides to reveal stains.
30s
Consult troubleshooting section to identify issues in slides, as most may be identified by this point.
Cover slipping
After several hours, use an eyedropper to squeeze Permount along the length of the slide.
Carefully place a coverslip in one smooth motion to spread mounting medium,
Take care to avoid air bubbles, especially over the sample.
Place slides in cardboard slide storage holders to aid spread of Permount across coverslip.
Tip one of the edges of the holder up on its edge to further improve preservation.
Acknowledgements
National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant ID: UC2AR082196