The ST samples were subjected to standard overnight fixation in 10% neutral-buffered formalin followed by paraffin embedding and were cut into 10 μm-thick slices. The slides were then deparaffinized in xylene and ethanol, incubated with Proteinase-K for 10 min at 37 °C, dehydrated with gradient ethanol, and hybridized with 1 nM LNA™ U6 snRNA probe and 50 nM double-DOG LNA™ miRNA probe complementary to miR-23b and LNA™ scrambled miRNA probe, respectively (all the probes were purchased from Exiqon, Demnark). After incubating with and removing the blocking solution, the slides were sequentially incubated with anti-DIG reagent and AP substrate (all were procured from Roche, Mannheim, Germany) protecting from light. Finally, KTBT buffer was added to stop the reaction. Nuclear Fast Red (Solarbio, China) was used for counterstaining of the nucleus. The slides were finally observed under light microscopy.
Nuclear fast red
Manufactured by Solarbio
Sourced in China
Nuclear Fast Red is a staining reagent used in histological and cytological applications. It is a synthetic dye that selectively stains the nuclei of cells, providing a red coloration. The dye binds to the DNA and RNA within the cell nucleus, allowing for the visualization and differentiation of cellular structures. Nuclear Fast Red is commonly used in routine tissue staining procedures as a counterstain or as part of a multi-step staining protocol.
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Lab products found in correlation
4 protocols using nuclear fast red
1
In Situ Hybridization of miR-23b in Tissue
2
Perls' Prussian Blue Staining for Iron Quantification
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Perls’ Prussian blue staining was used to analyze iron deposition in neointima. The cross-sections of ligated carotid arteries were fixed with 4% (v/v) formaldehyde (G1101, Servicebio) and washed with PBS 3 times. Sections were incubated in Perl’s solution (G1422, Solarbio) for 30 min, and deionized water was used to rinse three times. After incubation with Nuclear Fast Red (G1422, Solarbio) for 7.5 min, the sections were set in 75%, 85%, 95%, and 100% ethanol for rapid gradient dehydration. Iron+ cells were counted at ×200 magnification.
3
Histological Assessment of Kidney and Aortic Tissues
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Rat kidney and aortic tissues were fixed in 10% neutral formalin solution and embedded in paraffin. Then, paraffin-embedded tissues were cut into 4 μm sections, which were deparaffinized and rehydrated. The renal tissues were stained with hematoxylin and eosin to evaluate the severity of renal lesions under light microscopy (Olympus, Japan).
Aortic tissues were stained with von Kossa and Alizarin red staining for the evaluation of calcification, separately. For von Kossa staining, the sections were covered with 5% silver nitrate (Shanghai Yuanye Biotech., China) for 30 min and then exposed to ultraviolet light for 60 min. After washing, the sections were covered with 5% sodium thiosulfate and incubated for 5 min. The sections were washed again and counterstained with nuclear fast red (Solarbio, Beijing, China) to visualize the nuclei. For Alizarin red staining, after deparaffinization and washing, the tissues were stained with 2% Alizarin red solution for 30 min at room temperature with gentle rotation. The sections were mounted on glass slides, and images were observed under a light microscope (Nikon, Japan).
Aortic tissues were stained with von Kossa and Alizarin red staining for the evaluation of calcification, separately. For von Kossa staining, the sections were covered with 5% silver nitrate (Shanghai Yuanye Biotech., China) for 30 min and then exposed to ultraviolet light for 60 min. After washing, the sections were covered with 5% sodium thiosulfate and incubated for 5 min. The sections were washed again and counterstained with nuclear fast red (Solarbio, Beijing, China) to visualize the nuclei. For Alizarin red staining, after deparaffinization and washing, the tissues were stained with 2% Alizarin red solution for 30 min at room temperature with gentle rotation. The sections were mounted on glass slides, and images were observed under a light microscope (Nikon, Japan).
4
Histological Analysis of Cartilage Degeneration
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The human cartilage or mouse articular joint specimens were fixed in 4% paraformaldehyde, dehydrated, decalcified in 0.5 M EDTA, embedded in paraffin, and sectioned at 3 μm. The sections were deparaffinized in xylene and hydrated with graded ethanol. For safranin O/fast green staining, the sections were first stained with 1% fast green (Sigma-Aldrich) for 3 to 5 min, rinsed with 1% acetic acid for 10 s, and then stained with 1% safranin O (Sigma-Aldrich) for 3 to 5 min. For alcian blue staining, the sections were stained with 1% alcian blue (Solarbio) for 30 min and then stained with nuclear fast red (Solarbio) for 10 min. Cartilage destruction was evaluated by OARSI grade (47 ), Mankin score (48 (link)), and osteophyte formation score (57 ) consisting of two domains: size and maturity. For immunohistochemistry, the sections were deparaffinized in xylene and hydrated with graded ethanol. Next, the sections were incubated with 3% H2O2 and 5% bovine serum albumin (BSA) at room temperature and with the indicated antibodies overnight. Subsequently, a secondary antibody conjugated with horseradish peroxidase (HRP) was added to the sections, followed by 3,3'-diaminobenzidine (DAB) staining (Sigma-Aldrich). The sections were finally stained with hematoxylin (Beyotime), washed and scanned using a KFBIO scan and analysis system (KFBIO, Zhejiang, China).
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