Liver specimens were initially fixed thoroughly using formalin (F8775, Sigma–Aldrich), followed by paraffin embedding and sectioning. These sections were then deparaffinized and subjected to proteinase K treatment (ST532, Beyotime) for 15 minutes. The slides were subsequently rinsed three times in PBS, followed by the addition of 50 μL of TUNEL reaction mix (C1088, Beyotime) and incubation for 60 minutes. Then, the nuclei were stained with DAPI (D212, Dojindo). Finally, the stained sections were imaged for every sample (200× magnification) using a fluorescence microscope (IX81-FV1000, Olympus).
St532
Manufactured by Beyotime
Sourced in China
The ST532 is a laboratory equipment product. It is designed to perform specific functions in a laboratory setting. The core function of the ST532 is to facilitate various laboratory processes and procedures.
Automatically generated - may contain errors
Lab products found in correlation
F8775, by Merck Group (3 mentions)
Fv1000 ix81, by Olympus (1 mentions)
Tunel reaction mix, by Beyotime (1 mentions)
C1088, by Beyotime (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Tunel reaction mixture, by Beyotime (1 mentions)
Bafilomycin a1, by MedChemExpress (1 mentions)
Dynasore, by MedChemExpress (1 mentions)
Zli 9017, by ZSGB-BIO (1 mentions)
Tunel pod, by Roche (1 mentions)
Tunel reaction mixture, by Roche (1 mentions)
Pv 6001, by ZSGB-BIO (1 mentions)
4 protocols using st532
1
Liver Apoptosis Quantification Protocol
2
TUNEL Assay for Apoptosis Detection
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The sample was fixed in formalin (F8775, Sigma-Aldrich, Shanghai, China) and cut into 3 μm slice after paraffin embedding. Tissue sections were pre-treated proteinase K working solution (20 μg/mL in 10 mM Tris/HCl, pH 7.4-8, ST532, Beyotime, Shanghai, China) for 15 min at room temperature after the tissue sections were dewaxed and rehydrated. PBS rinse the slides 3 times. Add 50 μL of TUNEL reaction mixture (C1088, Beyotime, Shanghai, China) to the sample and incubate for 60 min in a dark and humidified chamber at 37 °C. After three times PBS rinse, DAPI (D212, 1:5000, Dojindo, Beijing, China) was used to stain the nuclei. The immunofluorescent images were captured under a fluorescence microscope (Olympus, Tokyo, Japan). The image was then quantified and analyzed by Image J (version 1.8.0).
3
Investigating NETs Interaction with BMDMs
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BMDMs were seeded in confocal-well at a density of 1×106/ well, followed by addition of 50 µg of mice NETs and further incubation for 1, 2 or 6 hours. To investigate the NETs location, BMDMs were pretreated with 1 µg/mL Dynasore (HY-15304, MedChemExpress, USA),26 27 (link) or 40 µg/mL of Bafilomycin-A1 (HY-100558, MedChemExpress, USA) for 30 min in complete media.28 (link) To investigate the role of Rab5a or other GTPases, BMDMs were pretreated with siRNA transfection, detailed in the methods of siRNA Knockdown. To investigate role of NETs containing proteins, NETs were pretreated with a proteinase K (40 µg /mL, ST532, Beyotime, Jiangsu, China) and heat-inactivated (65°C for 10 min).5 29 (link) To investigate the role of Neutrophil elastase (NE), BMDMs were treated with 50 nM recombinant NE (4517-SE-010, R&D Systems, Minnesota, USA) for 6 hours.30 (link) Supernatants were then harvested to detect the levels of IL-6 and TNF-α. BMDMs were washed twice with phosphate-buffered saline (PBS), then either lysed or fixed for additional experiments.
4
TUNEL Assay for Apoptosis Detection
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The specimens were fixed in formalin (Sigma-Aldrich, F8775) and embedded in paraffin before being cut into 3 μm slices. The tissue sections were pretreated with 3% H2O2 (ZSGB-BIO, PV-6001) at room temperature for 10 min and Proteinase K working solution (Beyotime, ST532, 20 μg/mL in 10 mM Tris/HCl, pH 7.4–8) at room temperature for 15 min after dewaxing, rehydration and antigen retrieval. Add 50 μL TUNEL reaction mixture (Roche, 11684817910) on sample and incubate 60 min at 37°C in a humidified chamber in the dark. Rinse slide 3 times with PBS, add 50 μL TUNEL-POD (Roche, 11772465001) on sample and incubate slide in a humidified chamber for 30 min at 37°C. Rinse slide 3 times with PBS and incubate with DAB (ZSGB-BIO, ZLI-9017). Nuclei were stained in hematoxylin for 3 s. The histological and immunohistochemical images were observed and captured under a light microscope (Olympus, Tokyo, Japan).
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