The spleen paraffin sections were deparaffinized in graded xylene and ethanol, followed by staining with hematoxylin (Beyotime) for 30 min and eosin (Beyotime) for 3 min. Subsequently, the sections were dehydrated using a series of graded ethanol solutions and xylene. After dehydration, a coverslip was applied using resinene (Solarbio).
Resinene
Manufactured by Solarbio
Sourced in Germany
Resinene is a laboratory equipment product manufactured by Solarbio. It is a specialized resin-based material designed for use in various scientific applications. Resinene's core function is to provide a durable and stable platform for the processing and handling of samples and materials in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Beyotime (2 mentions)
Inverted phase contrast microscope, by Zeiss (1 mentions)
Safranin o, by Merck Group (1 mentions)
Anti mtor, by Affinity Biosciences (1 mentions)
Anti cyclin d1, by Affinity Biosciences (1 mentions)
Anti cdk4, by Affinity Biosciences (1 mentions)
Anti cdk6, by Affinity Biosciences (1 mentions)
Dab peroxidase substrate kit, by Solarbio (1 mentions)
Goat serum, by Zhongshan Jinqiao Biotechnology (1 mentions)
3 protocols using resinene
1
Spleen Tissue Histological Analysis
2
Chondroitin Sulfate Production Assay
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Following 2, 4 and 6 days of culture in 24-well plates as described above, the cells on the cover slips were washed three times with PBS and fixed with 95% ethanol for 30 min. The cells on the cover slips were then stained with 0.1% Safranin O (Sigma-Aldrich; Merck Millipore) for 5 min, followed by washing with tap water for 3 min. Following sealing with resinene (Beijing Solarbio Science and Technology Co., Ltd.), the cells were imaged using an inverted phase contrast microscope (Zeiss AG, Oberkochen, Germany).
In another experiment, cells incubated with baicalin in 6-well plates for 2, 4 or 6 days as described above were washed three times with PBS and subjected to enzymolysis with 0.25% trypsin. Subsequently, cells were resuspended in 1 ml PBS containing 0.1 µg proteinase K at 60°C for 6 h. The production of glycosaminoglycans (GAGs) was calculated by assessing the absorbance value of the cell enzyme solution with 1,9-dimethylmethylene blue using a Multiskan GO microplate reader at 525 nm and comparing with the standard curve of chondroitin sulfate (23 (link)). The production of GAGs was standardized to the DNA content of the cells, which indicated the activity of cell replication in the presence of baicalin at different concentrations.
In another experiment, cells incubated with baicalin in 6-well plates for 2, 4 or 6 days as described above were washed three times with PBS and subjected to enzymolysis with 0.25% trypsin. Subsequently, cells were resuspended in 1 ml PBS containing 0.1 µg proteinase K at 60°C for 6 h. The production of glycosaminoglycans (GAGs) was calculated by assessing the absorbance value of the cell enzyme solution with 1,9-dimethylmethylene blue using a Multiskan GO microplate reader at 525 nm and comparing with the standard curve of chondroitin sulfate (23 (link)). The production of GAGs was standardized to the DNA content of the cells, which indicated the activity of cell replication in the presence of baicalin at different concentrations.
3
Immunohistochemical Analysis of Spleen
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First, the spleen paraffin sections were deparaffinized in xylene and graded ethanol. To block endogenous peroxidase activity, the sections were incubated with 10% hydrogen peroxide. For antigen retrieval, the sections were heated in 2% EDTA solution for 15 min, and allowed to cool for 2 h at room temperature. After washing with PBS, the slides were blocked with goat serum (Zhongshanjinqiao Biotechnology Co., Ltd., China) for 15 min, and then incubated with the diluted antibodies at 4℃ overnight. On the next day, the sections were incubated with corresponding secondary antibodies at room temperature for 15 min. The sections were then labeled with horseradish peroxidase, and the signals were detected using the DAB Peroxidase Substrate Kit (Solarbio). After staining with hematoxylin (Beyotime) for 30 s, the slides were dehydrated in graded ethanol and xylene. Finally, the sections were sealed with coverslips by resinene (Solarbio). The following primary antibodies were used for the assay: anti-GS (Affinity), anti-mTOR (Affinity), anti-Cyclin D1 (Affinity), anti-CDK4 (Affinity), anti-CDK6 (Affinity).
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