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Safranin o fast green staining

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Safranin O/Fast Green staining is a laboratory staining technique used for the visualization and differentiation of cellular structures. It serves as a contrast stain, enhancing the visibility of various components within a sample. The Safranin O dye stains nucleic acids, while the Fast Green dye stains cytoplasmic and extracellular structures.

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Aggrecan, by Abcam (1 mentions) Ab84594, by Abcam (1 mentions) Ab3773, by Abcam (1 mentions) Ab58703, by Abcam (1 mentions) Ab34712, by Abcam (1 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) Fluoview fv1000 confocal microscope, by Olympus (1 mentions) Normal saline, by Merck Group (1 mentions) Eclipse c2 plus, by Nikon (1 mentions) Cm1520, by Leica Biosystems (1 mentions) Paraformaldehyde (pfa), by Biosesang (1 mentions) Calci clear rapid, by National Diagnostics (1 mentions) Aperio scanscope cs, by Leica Biosystems (1 mentions)

8 protocols using safranin o fast green staining

1

Quantifying Cartilage Degeneration in Joints

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Cartilage structure was evaluated using Safranin O/Fast Green staining (Sigma) to visualize cartilage and bone36 (link). Joint sections were prepared and imaged as described for ligament. Stained articular surfaces (6–8 images/rat) were scored by two blinded graders using a modified Mankin scale to assess cartilage degradation38 , based on cellular and background staining, chondrocyte arrangement, and structural surface condition, with scores ranging from normal (0) to maximally degenerate (10)38 , and averaged across all images for each rat.
Joint space and cartilage width were measured using FIJI (NIH; Bethesda, MD) to evaluate joint space narrowing and cartilage degradation5 (link). Joint space width was quantified as the perpendicular distance between the articular surfaces; cartilage width was defined by the length of the Safranin O staining perpendicular to the subchondral bone (Fig. 3a). Both measurements were made in triplicate for each image (3–4 images/rat) from the lateral to medial end of the joint and averaged.
Ita M.E., Ghimire P., Welch R.L., Troche H.R, & Winkelstein B.A. (2020). Intra-articular collagenase in the spinal facet joint induces pain, DRG neuron dysregulation and increased MMP-1 absent evidence of joint destruction. Scientific Reports, 10, 21965.
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2

Histological Analysis of Rat Knee Cartilage

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The cartilage tissues of the rat knee joint were fixed with 4% paraformaldehyde and then decalcified with EDTA decalcifying solution (0.5 μM, pH = 8) for 15 days. The solution was changed every 3 days. Then, the tissues were embedded in paraffin and sliced into sections (4 μM thick). The pathological changes of the cartilage were observed by Safranin O/fast green staining (Sigma-Aldrich, St.Louis, MO, United States) in accordance with the manufacturer’s introduction.
Li J., Zhang Z., Qiu J, & Huang X. (2021). 8-Methoxypsoralen has Anti-inflammatory and Antioxidant Roles in Osteoarthritis Through SIRT1/NF-κB Pathway. Frontiers in Pharmacology, 12, 692424.
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3

Histological Analysis of Mouse Knee Joints

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The knee joints from mice were fixed with 4% paraformaldehyde for 24 h and then decalcified with 0.5 M ethylenediaminetetraacetic acid (EDTA) at pH 7.4 for 21 days. The specimens were embedded in paraffin and sectioned at 4 μm. For histological analysis, the samples were stained with hematoxylin and eosin (H&E) and safranin O-Fast Green staining (Sigma-Aldrich). For the immunohistochemistry analysis, we employed the following primary antibodies: A2M (ab58703, Abcam), MMP13 (ab84594, Abcam), and Cleaved Caspase-3 (#9661, CST). Sections were then stained with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). For immunofluorescence, the primary antibodies used were Collagen II (ab34712, Abcam), Aggrecan (ab3773, Abcam) and Alexa 594 dye-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The sections were mounted with medium containing DAPI and images were obtained using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan).
Liu X., Liu L., Zhang H., Shao Y., Chen Z., Feng X., Fang H., Zhao C., Pan J., Zhang H., Zeng C, & Cai D. (2019). MiR-146b accelerates osteoarthritis progression by targeting alpha-2-macroglobulin. Aging (Albany NY), 11(16), 6014-6028.
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4

Safranin O-Fast Green Staining of Cartilage Tissues

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Safranin O-Fast Green staining (Sigma-Aldrich; Merck KGaA) was performed to observe the morphology of cartilage tissues under a light microscope. Paraffin-embedded slices were dewaxed in xylene at 20˚C for 10 min and rehydrated with absolute alcohol for 5 min, 95% alcohol for 5 min and 80% alcohol for 5 min, prior to being washed with distilled water for 2 min. Sections were subsequently stained with haematoxylin at 20˚C for 3 min, before being washed three times with distilled water. Hydrochloric acid (1%) and ethanol were used for 15 sec at 20˚C to differentiate the slices. The slices were washed three times with distilled water and immersed in a 0.02% Fast Green solution at 20˚C for 3 min. The stained slices were washed in 1% glacial acetic acid and stained in 0.1% Safranin O at 20˚C for 3 min. The slices were sealed to observe the cellular matrix staining, tidemark and calcification of cartilage tissues under a light microscope (magnification, x100).
Zhao Q.H., Lin L.P., Guo Y.X., Zou R., Wang Z., Shi Z.P, & Fu-Qing L. (2020). Matrix metalloproteinase-13, NF-κB p65 and interleukin-1β are associated with the severity of knee osteoarthritis. Experimental and Therapeutic Medicine, 19(6), 3620-3626.
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5

Histological Analysis of Intervertebral Disc Degeneration

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The rats were perfused transcardially with 0.9% normal saline (Sigma-Aldrich, St. Louis, MO, USA) and 4% paraformaldehyde (Biosesang, Seong-nam, Korea) for histological staining and immunohistochemistry. The L4–5 and L5–6 discs were extracted, then post-fixed in 4% paraformaldehyde overnight at 4 °C. Tissues were immersed in 30% sucrose for 3 days and sectioned in 20-µm thicknesses using cryo-microtome (CM1520, Leica Biosystems, Nussloch, Germany). Safranin O/fast green staining (Sigma-Aldrich, St. Louis, MO, USA) was performed according to the manufacturer’s instructions on the L4–5 and L5–6 discs to confirm the degree of damage to the disc at 4 weeks. Stained sections were imaged using an inverted microscope (Eclipse C2 Plus, Nikon). The damage grade in the intervertebral discs was assessed using a histological grading scale based on four degenerative change categories (to assess the anulus fibrosus, the border between the anulus fibrosus and nucleus pulposus, the cellularity of the nucleus pulposus, and the matrix of the nucleus pulposus), with scores ranging from a normal disc with 4 points (1 point in each category) to a severely degenerated disc with 12 points (3 points in each category) [16 (link)].
Hong J.Y., Kim H., Jeon W.J., Lee J., Yeo C., Lee Y.J, & Ha I.H. (2022). Epigenetic Changes within the Annulus Fibrosus by DNA Methylation in Rat Intervertebral Disc Degeneration Model. Cells, 11(22), 3547.
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6

Preparation and Analysis of Rat Intervertebral Discs

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The spines of the rat tail, including the IVDs and adjacent vertebrae, were separated, fixed with 4% paraformaldehyde (PFA) for 48 h, and decalcified with 10% ethylenediaminetetraacetic acid (EDTA, pH 7.5) for 30 days after being washed with PBS. All IVDs were embedded in paraffin and then cut into 5-μm midsagittal sections. The sections were dewaxed and dehydrated according to the standard procedures, and HE staining and safranin O/fast green staining (Sigma Aldrich, St Louis, USA) were then performed. Images of the sections were captured with the Eclipse 80i fluorescence microscope.
Chen W., Deng Z., Zhu J., Yuan L., Li S., Zhang Y., Wu J., Huang Z., Qin T, & Ye W. (2023). Rosuvastatin suppresses TNF-α-induced matrix catabolism, pyroptosis and senescence via the HMGB1/NF-κB signaling pathway in nucleus pulposus cells: Role of rosuvastatin in alleviating intervertebral disc degeneration. Acta Biochimica et Biophysica Sinica, 55(5), 795-808.
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7

Histological Assessment of Ankle Joint

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The ankle joints were removed on day 35 and fixed for 24 h in 4% paraformaldehyde. The fixed tissues were decalcified in Calci-Clear Rapid (National Diagnostics, Atlanta, GA, USA), embedded in paraffin and sectioned (4 µm) using a microtome. Tissue sections were stained with hematoxylin and eosin and Safranin O/Fast Green staining (Sigma-Aldrich) to assess cartilage destruction. Bone destruction, vascular proliferation, synovial hyperplasia and inflammatory cell infiltration were assessed.
Han H.M., Hong S.H., Park H.S., Jung J.C., Kim J.S., Lee Y.T., Lee E.W., Choi Y.H., Kim B.W., Kim C.M, & Kang K.H. (2016). Protective effects of Fructus sophorae extract on collagen-induced arthritis in BALB/c mice. Experimental and Therapeutic Medicine, 13(1), 146-154.
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8

Histological Evaluation of Lateral Condyle

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The halves of the lateral condyles dedicated to histology were decalcified in formic acid/hydrochloric acid for about 1 month and then processed for paraffin embedding as indicated in a previous study [17 (link)]. Three slides of the central portion of the condyles were stained with Safranin O/Fast Green staining (Sigma-Aldrich, St. Louis, MO, USA) and the other three were immunostained for COLL II, using primary antibodies (anti-COLL II). Each slide was acquired with a Aperio ScanScope digital scanner (Aperio ScanScope CS, Aperio Technologies, Leica Biosystems, Milan, Italy). The three slides stained with Safranin O/Fast Green were histomorphometrically evaluated by measuring cartilage thickness (CT, µm) and fibrillation index (FI, %). In the three slides immunostained for COLL II content (%), the ratio between immunopositive stained areas and the total region of interest (ROI) areas was calculated.
Berni M., Veronesi F., Fini M., Giavaresi G, & Marchiori G. (2023). Relations between Structure/Composition and Mechanics in Osteoarthritic Regenerated Articular Tissue: A Machine Learning Approach. International Journal of Molecular Sciences, 24(17), 13374.
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