For hematoxylin-eosin (HE) staining, the bone callus samples were decalcified and embedded in paraffin. Then the samples were serially sectioned into 5-μm thickness. Next, the slices were stained with HE dye liquor. For Safranin O staining, the sections were p stained with Safranin O for 2 min (Solarbio, Beijing, China). Then the samples were treated in 95% ethanol for 3 s, treated twice in 100% ethanol for 2 min each, dewaxed twice in xylene for 10 min each. Lastly, these samples were mounted in neutral balsam (Sinopharm, Shanghai, China) and observed using a light microscope (Olympus, Tokyo, Japan).
Safranin o
Manufactured by Solarbio
Sourced in China
Safranin O is a dye commonly used in biological and medical applications. It is a red, crystalline powder that can be dissolved in water or alcohol. Safranin O is often used as a counterstain in microscopy techniques, such as Gram staining, to selectively stain certain cellular structures or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Toluidine blue, by Solarbio (4 mentions)
Alcian blue, by Solarbio (2 mentions)
Fast green fcf, by Solarbio (2 mentions)
Mmp13, by Abcam (2 mentions)
Ab34712, by Abcam (2 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (2 mentions)
Safranin o staining kit, by Solarbio (2 mentions)
Neutral buffered formalin, by Avantor (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Leica rm2145, by Leica camera (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
1 9 dimethylmethylene blue dmb, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Papain, by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Boster Bio (1 mentions)
Fast green, by Solarbio (1 mentions)
Freezing microtome, by Leica camera (1 mentions)
Sensys ccd camera, by Olympus (1 mentions)
Cm1850, by Leica camera (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
32 protocols using safranin o
1
Histological Analysis of Bone Callus
2
Safranin O Staining of Chondrocytes
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In order to detect the deposition of glycosaminoglycans (GAGs) from chondrocytes, safranin O staining kit (Beijing Solarbio Science & Technology Co., Ltd.) was used to perform the safranin O staining assay according to the manufacturer's protocol. Cells were harvested and fixed using 95% ethanol for 30 min at room temperature, followed by washing three times with PBS. Next, 0.1% safranin O (Beijing Solarbio Science & Technology Co., Ltd.) solution was used to stain the cells for 10–15 min at room temperature. Staining was observed using a light microscope (magnification, ×100; Olympus Corporation) and images were captured.
3
Rabbit Cartilage Histological Analysis
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Following sacrifice, the lateral femoral condyles of the rabbits were removed and fixed in 10% neutral buffered formalin (pH 7.4; VWR International, Radnor, PA, USA) for 48 h and decalcified with 20% EDTA (Gibco). This was followed by dehydration through a series of increasing concentrations of ethanol. The samples were paraffin-embedded, and 5-μm-thick microsections [created using a microtome (Leica RM2145; Leica, Wetzlar, Germany)] were stained with Safranin-O (Cat. no. G2540; Solarbio). The grading of Safranin-O staining was evaluated by blinded individuals according to the Mankin scoring system (28 (link)).
4
Chondrocyte Differentiation Evaluation
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Sodium alginate was purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). Chitosan was obtained from Golden-Shell Biochemical Co., Ltd. (Zhejiang, China). Cysteine, hematoxylin and eosin (H&E) staining kit, papain, and safranin-O were kindly purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Masson stain kit was obtained from Jiancheng Technology Co., Ltd. (Nanjing, China) C5.18 cells, acridine orange/ethidium bromide (AO/EB) and klcian blue staining Kit were provided by Syagen Biosciences Inc. (Guangzhou, China). Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 1,9-dimethylmethylene blue (DMB) was obtained from Sigma Co., Ltd. (St. Louis, MO, USA). Rat collagen type-II (Col-II) ELISA kit was purchased from Cusabio Biotech Co., Ltd. (Wuhan, China). Basic fibroblast growth factor (bFGF) was purchased from PeproTech Inc. (Rocky Hill, CT, USA).
5
Paraformaldehyde-Fixed Tissue Sectioning and Staining
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The samples were fixed in 4% paraformaldehyde (BOSTER, China) for 72 h. After dehydration for 24 h, the samples were embedded in optimal cutting temperature compound (OCT, sakura, United States), and then performed frozen sectioning (LEICA, Germany) with a slice thickness of 8 μm. Random sections from each group were stained with HE, Safranin-O and Fast Green (Solarbio, G1371, China). Detailed operation was conducted according to the product instructions. Briefly, the frozen sections were returned to room temperature, and rinsed with tap water and then added with working solution for staining. Finally, the sections were dehydrated in alcohol and sealed with resinene. After histological staining, images were taken under a ×20 or ×40 objective with a resolution of 4,080 × 3,072. The image was taken by a light microscope (Olympus, BX53, Japan).
6
Cartilage Extracellular Matrix Staining
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Rat chondrocytes were seeded into a 24-well plate and allowed to grow until they reached 75% confluency. Afterward, the cells were gently washed three times with PBS and fixed with 4% paraformaldehyde for 15 min, followed by another round of PBS washing. Subsequently, carefully add the respective staining solutions, including toluidine blue, Alcian blue, and safranin O (solarbio, China), to the wells, and let them incubate undisturbed at room temperature for 30 min. Finally, remove the excess dye by washing with PBS, and observe the stained cells under an ordinary light microscope.
7
Histological Assessment of Knee Osteoarthritis
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The femoral condyles were collected from the sacrificed rats, fixed with 4% paraformaldehyde for 48 h, decalcified with 12.5% ethylenediaminetetraacetic acid for 4 weeks, and dehydrated with 30% sucrose solution for 72 h. The articular cartilage was then isolated, frozen, and embedded in OCT compound (Tissue-Tek, Tokyo, Japan). Finally, sections of 7 µm thick were cut in the sagittal plane with a freezing microtome (Leica, Heidelberger, Germany). The frozen sections were fixed again with acetone solution for 10 min, and then stained with hematoxylin and eosin (HE), toluidine blue, and safranin O (Solarbio, Shanghai, China), respectively. The Osteoarthritis Research Society International (OARSI) scoring system was used to determine the extent of cartilage deterioration and the severity of knee OA, with scores ranging from 0 (normal) to 6 (>80% loss of cartilage). The modified Mankin criteria were used to assess cartilage damage, with scores ranging from 0 (normal) to 24 (the most severe OA)28 (link). The evaluations were performed independently by two blinded observers and the scores were evaluated in OA, BMSC, and BMSC + CCM groups.
8
Microscopic Analysis of Internode Development
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At the jointing-booting stage, the second internode stems of DPW and HPW treated with different photoperiods, under growing seasons and sprayed with phytohormones were fixed in Carnoy's fixative I (3 parts of 95% ethanol to 1 part of glacial acetic acid). Sections (cross and longitudinal) with 10-μm-thick were cut on a cryostat microtome (Leica, CM1850, UV) with 1% agarose gel. After removing agarose gel using QIAquick Gel Extraction Kit (Qiagen, Cat. #: 28706, USA), the sections were stained with 1% safranin O (Solarbio) in water for 3 hours, rinsed three times in 85% ethanol for 10 minutes, and then counterstained with an alcoholic solution of fast green FCF (Solarbio, Cat. #: 907A035) (1 mg fast green FCF in 200 ml 50% ethanol) for 20 seconds. Images were captured using a Photometrics SenSys CCD camera on an Olympus BX-51 microscope (Olympus, Tokyo, Japan).
9
Histological Analysis of Articular Cartilage
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Cartilage specimens were fixed with 4% paraformaldehyde (Solarbio Beijing, China) for 48 h at 4°C, decalcified in 20% EDTA solution (Merck, Germany), dehydrated through graded ethanol solutions, embedded in paraffin, and sliced into 5-μm sections. Sections were stained with hematoxylin and eosin (H&E; Solarbio), Safranin O (Solarbio), and toluidine blue (Solarbio), and then sealed with neutral gum. Finally, the specimens were assessed under a light microscopy (Nikon, Japan). Five slices of each articular cartilage specimen were selected, and KOA was evaluated by measuring the Mankin score(Van Der Sluijs et al., 1992 (link)).
10
Saikosaponin D Pharmacological Evaluation
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Saikosaponin D (purity >98%) was purchased from MCE (New Jersey, United States). DMSO (Meilunbio, Dalian, China) was used to dissolve Saikosaponin D and diluted in the cell culture until DMSO <0.1%. Recombinant mouse IL-1β was obtained from Peprotech (United States). Safranin-O and Fast Green and tartrate-resistant acid phosphatase staining (TRAP) were acquired from Solarbio (Beijing, China). Primary antibodies were applied in this study: antibodies against P65 (#8242), p-P65 (#3033), IκBα (#4814), p-IκBα (#2859), ERK (#4695), p-ERK (#4370), JNK (#9252), p-JNK (#9255), P38 (#8690), and p-P38 (#4511S) were acquired from CST (1:1,000, Cambridge, MA, United States). Anti-iNOS (1:1,000, ab178945), anti-COX2 (1:4,000, ab179800), anti-Aggrecan (1:1,000, ab3778), and anti-ADAMTS5 (1:250, ab41037) were purchased from Abcam (Cambridge, United Kingdom). Antibodies against MMP13 (1:2000, 18165-1-AP), Collagen II (1:1,000, 28459-1-AP), Nrf2 (1:2000, 66504-1-Ig), HO-1 (1:1,500, 27282-1-AP), Lamin B (1:10000, 66095-1-Ig), and Actin β (1:8,000, 66009-1-Ig) were obtained from Proteintech (Wuhan, China). Goat anti-mouse and goat anti-rabbit were acquired from Thermo Fisher (United States).
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