After eight weeks of MON treatment, the animals were euthanized and the right knee tissue was fixed with 4% PFA and then decalcified with ethylenediaminetetraacetic acid (EDTA). After decalcification, the tissues were dehydrated through an ethanol gradient (70%, 80%, 95–100%), cleared with xylene, and embedded in paraffin. The wax blocks were sliced into 5 µm-thick sections that were baked overnight in an oven at 37 °C. The sections were dewaxed and stained 0. 2% Safranin O solution for 15 min, rinsed with distilled water, counterstained with 0. 2% Fast Green solution for 5 min (Sigma-Aldrich, USA), and sealed. The structural changes in the cartilage tissues were evaluated in a blinded manner according to the Osteoarthritis Research Society International (OARSI) scoring system [6 (link)]. Hematoxylin–eosin (HE) staining was performed as per standard protocols, and the inflammation around the lesions was scored. The slides were observed using the Olympus IX73 microscope.
Fast green solution
Manufactured by Merck Group
Sourced in United States
Fast Green solution is a laboratory reagent used for staining and visualization purposes. It serves as a colorant in various biological and chemical applications. The solution contains the dye Fast Green FCF, which provides a green coloration. This product is intended for use in controlled laboratory settings by trained personnel.
Automatically generated - may contain errors
Lab products found in correlation
Safranin o solution, by Merck Group (19 mentions)
Weigert s iron hematoxylin solution, by Merck Group (4 mentions)
Antibodies for hoxa1, by Abcam (2 mentions)
Optical microscope, by Nikon (2 mentions)
Pentobarbital sodium, by Merck Group (2 mentions)
Weigert s iron hematoxylin, by Merck Group (2 mentions)
Xylene, by Merck Group (2 mentions)
Ix73 microscope, by Olympus (1 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
Alcian blue solution, by Merck Group (1 mentions)
Triton x 100 solution, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Hematoxylin, by Wuhan Servicebio Technology (1 mentions)
Dako envision kit, by Agilent Technologies (1 mentions)
Hematoxylin, by Agilent Technologies (1 mentions)
Cx41 microscope, by Olympus (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Colorview camera, by Olympus (1 mentions)
Haematoxylin, by Thermo Fisher Scientific (1 mentions)
33 protocols using fast green solution
1
Histological Analysis of Osteoarthritis in Mice
2
In vivo CHD6 overexpression in chicken embryos
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For in vivo CHD6 overexpression, electroporation of chicken embryos37 (link) was performed on eggs from stage HH9-10 that were windowed and had the extraembryonic membrane partially removed. A vector containing mutant or wild type CHD6 ORFs (Ka0717_pPb-hCMV-cHA-IRES Venus-CHD6m and Ka0717_pPb-hCMV-cHA-IRES Venus-CHD6m; this paper) were first mixed 9:1 with the plasmid encoding the transposition transactivator, and this mixture then mixed 2:1 with Fast Green solution (Sigma) and microinjected into the neural tubes of the embryos. The neural tube was then electroporated with 5x square pulses of 20 V/20 msec using the Intracel TSS20 Ovodyne electroporator, eggs were resealed with tape and re-incubated for 24 h, before tape was removed and 10 µl of 2 µg/ml doxycycline were added at the site of electroporation and eggs were sealed again and re-incubated until stage HH20, where heart dissections also took place. CHD6 overexpression was confirmed in transgenic embryos on the basis of GFP signal under an Olympus SZX16 stereomicroscope with an EXFO X-cite series 120PC Q apparatus. Under German law, experiments on fertilized eggs performed up until stage HH20 do not require animal ethics approval.
3
Histological Analysis of Osteoarthritis in Human Knee Joints
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Human knee joint tissues after were embedded in paraffin wax and sectioned to 5 μm. Then, sections were incubated in Fast Green solution (Sigma, cat. no. F7252, USA) for 3 min. Following thorough washing with tap water, the sections were incubated for 10 s with Safranin‐O solution (Sigma, cat. no. S8884, USA). For Alcian blue staining, cells were incubated in an Alcian blue solution (Sigma, cat. no. 33864‐99‐2, USA) for 1 h. Osteoarthritis Research Society International (OARSI) grading system (0–6) was used to evaluate OA severity.
Sections and cells permeabilized by Triton X‐100 solution (Sigma, cat. no. T8787, USA), followed by incubation with 5% BSA for 2 h. After that, the sections and cells were incubated overnight at 4°C with primary antibodies (TableS5) . After incubation, the primary antibodies were washed three times in PBS to remove unbound antibodies. It was then incubated with Alexa 488–conjugated goat anti‐mouse secondary antibodies (1:500, Beyotime, cat. no. A0428, China) and Alexa 555‐conjugated donkey anti‐rabbit secondary antibodies (1:500, Beyotime, China). DAPI (Invitrogen) was used as a stain for nuclei. An inverted fluorescence microscope (Leica) was used to observe cell samples and scan sections using a fluorescence microscope (Leica).
Sections and cells permeabilized by Triton X‐100 solution (Sigma, cat. no. T8787, USA), followed by incubation with 5% BSA for 2 h. After that, the sections and cells were incubated overnight at 4°C with primary antibodies (Table
4
Histological Analysis of Fractured Tibia
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For histological analysis, the fractured tibia was decalcified, paraffin was embedded, and the tibia was cut into 10 μm sections. Paraffin sections were rehydrated and stained with hematoxylin (Servicebio, Wuhan, China) for 1 min. After rinsing with water, the sections were stained with 0.2% Fast Green solution (Sigma Aldrich, USA), then incubated in 1% acetic acid for 12 s. Next, the sections were stained with 0.1% Safranin O solution (Sigma Aldrich, USA) for 3 min. The slides were washed, dehydrated, and mounted with resin mounting solution before taking images.
5
Analyzing NP Extracellular Matrix in 3D Grafts
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In order to analyze the production of NP extracellular matrix components in 3D transplants, the grafts were dissected into 4 parts, embedded in TissueTek (Sakura Finetek, Staufen, Germany), frozen in liquid nitrogen and stored at -80°C until sectioning. Cryosections with a thickness of 6 μm were prepared. To demonstrate proteoglycan formation, Safranin O staining was conducted. The samples were incubated for 30 min with a 0.7% Safranin O solution (Sigma-Aldrich) and subsequently counterstained with a 0.2% Fast green solution (Sigma-Aldrich). For the immunochemical detection of collagen type II, a primary monoclonal rabbit-anti-human antibody (Acris Antibodies, Herford, Germany) was used. The detection was performed using the DAKO EnVision Kit (DAKO, Hamburg, Germany) according to the manufacturer’s protocol. In brief, the cryosected transplants were incubated with primary antibody solution for 40 min. The secondary antibody (horseradish peroxidase labeled goat-anti-rabbit antibodies) solution was also applied for 40 min. Finally, the cryosected transplants were incubated with the substrate AEC for 10 min and then counterstained with hematoxylin (DAKO) for 10 minutes. Pictures of both stainings were taken using an Olympus CX41 microscope and an Olympus Colorview camera (Olympus Soft Imaging Solutions GmbH, Hamburg, Germany).
6
Cartilage Destruction Evaluation and Apoptosis Detection
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Cartilage specimens were fixed in 4% paraformaldehyde for paraffin embedding. Each paraffin-embedded cartilage sample was sectioned at 5 μm, and every 10th section was stained with 0.1% safranin O solution and 0.001% Fast Green solution (Sigma-Aldrich, St. Louis, MO, USA). Cartilage destruction of mouse knee joints was scored by two observers blinded to group-identifying information using the OARSI grading system.28 (link) TUNEL staining was performed using the In Situ Cell Death Detection kit, POD (Sigma-Aldrich), according to the manufacturer’s protocol. For IHC analysis, the sections were incubated at 4°C with antibodies for HOXA1 (Abcam, Cambridge, UK) overnight and for 2 h at room temperature with secondary antibodies (Beyotime Institute of Biotechnology, Shanghai, China). The number of positively stained cells on the entire articular surface (including the femoral condyle and tibial plateau area) per specimen was counted, and the percentage of positive cells was calculated.29 (link)
7
Histological Evaluation of Knee Cartilage
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After deparaffinization, the right knee joint was reacted with Weigert's Iron Hematoxylin solution (Sigma, USA) for 10 min and stained with 0.001% fast green solution (Sigma, USA) for 5 min. The knee joint tissue was then reacted with 1% acetate solution for 10 s and stained with 0.1% safranin O solution (Sigma, USA) for 5 min. The tissue was then dehydrated and observed under an optical microscope (Nikon, Japan).
8
Histologic Evaluation of Osteoarthritis in Mice
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After surgery, mice that received different treatments were kept separately, and each treatment group had two cages (3 mice in a 400 square inch cage). At 8 weeks post-surgery, cartilage specimens were fixed in 4% paraformaldehyde for paraffin embedding. Each paraffin-embedded cartilage sample was sectioned at 5 μm, and every tenth section was stained with 0.1% safranin O solution and 0.001% Fast Green solution (Sigma‒Aldrich, St. Louis, MO, USA). For simple histologic scoring of OA in the mouse, we used an approved 0–6 subjective scoring system [22 (link)]. Histologic scores were evaluated in a blinded manner according to a grading scale (0 for normal cartilage, 0.5–4 for moderately degenerated cartilage, and 5–6 for severely degenerated cartilage).
9
Histological Assessment of Intervertebral Disc Degeneration
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To measure the degree of IVDD, all rats were sacrificed at 8 weeks after surgery. After tissue collection, tissue sections (5 µm) were carefully cut. In addition, the disc tissue sample sides were carefully stained by Safranin O fast green (SO), Haematoxylin and eosin (H.E) and Alcian Blue (A.B), according to the manufacturer’s protocols. For SO staining, deparaffinized sections were stained with SO solution (Sigma-Aldrich) and subsequently counterstained with 0.2% fast-green solution (Sigma-Aldrich). For HE staining, deparaffinized sections were stained with Haematoxylin (Thermo Scientific, UK) for 7 min and counterstained in Eosin (Thermo Scientific, UK) for 1 min. A.B staining was performed using Alcian Blue solution (0.1% A. B 8GX in 0.1 N HCl) for 30 min at room temperature. H. E staining was performed to evaluate the morphological changes of NPCs. The SO staining and A. B staining were performed to detect the cellularity and morphology of NP tissues examined by other three histology analysts in a blinded manner by a light microscope and carefully evaluated by using a grading scale, as described previously [34] (link). Therefore, the histolopathological scores were 5 for the normal disc, 6–11 for the moderate degenerated disc and 12–15 for the severe degenerated disc. In the end, images were scrupulously captured using a light microscope (Nikon, Japan).
10
Histological Staining and Scoring of Osteoarthritis in Rats
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Deparaffinized sections (3–5 μm) were Haematoxylin Eosin (HE) stained using a standard protocol. For Alcian Blue (AB) staining, the sections were incubated for 3 minutes in 1 % acetic acid and then stained 15 minutes in 1 % AB (Carl Roth GmbH). Subsequently, they were rinsed in 3 % acetic acid and cell nuclei counterstained using nuclear fast red aluminium sulfate solution (Carl Roth GmbH) for 5 minutes. For the safranin-O staining the slides were incubated for 10 minutes in a Weigert’s iron hematoxylin (Carl Roth GmbH) and afterwards rinsed in running tap water for 10 minutes. Subsequently, the sections were stained in 0.001 % fast green solution (Sigma-Aldrich) for 5 minutes. After rinsing them briefly in 1 % acetic acid the slides were stained in 0.1 % safranin-O solution (Merck KGaA) for up to 5 minutes. Dehydrated sections were mounted with Entellan mounting medium (Merck KGaA). The stained sections were further analyzed by applying score systems by two observers. To consider species-dependent peculiarities in rats two scoring systems for OA were used, the well-established Mankin Score, which was originally designed for human cartilage and the score system adapted by Glasson et al., [10 (link)] suitable for the very thin rodent joint cartilage. The scoring system according to Krenn et al., [11 (link)] was applied to classify synovitis.
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