For ethynyl deoxyuridine (EdU) assay, P7 CKO and WT newborn mice were injected intraperitoneally with EdU (ST067, Beyotime; 20 mg/g) and euthanized 4 h later. As described previously, the spine tissues were fixed, and 6-μm frozen sections were prepared. EdU was detected using the BeyoClick™ Cell Proliferation Kit with Alexa Fluor 555 (C0075S, Beyotime) following the manufacturer's instructions. For the TUNEL assay, we fixed spine tissues of P7 CKO and WT mice and then prepared 6-μm frozen slices. Following the manufacturer's manual, we detected apoptotic chondrocytes in the vertebra using the TdT-mediated dUTP Nick-End Labelling kit (C1088, Beyotime). Briefly, slices were incubated with 20 μg/mL proteinase K for 30 min at 37 °C and then transferred to TUNEL detective solution for 1 h at 37 °C, followed by counterstaining and mounting with DAPI (Sigma, SLCJ8103). Fluorescence microscopy (Olympus, BX63, Japan) was used to capture the immunofluorescence staining images.
St067
Manufactured by Beyotime
Sourced in China
The ST067 is a laboratory equipment designed for precision temperature regulation. It features advanced temperature control technology to maintain a stable and uniform temperature environment within the designated area.
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Lab products found in correlation
5 protocols using st067
1
Quantifying Chondrocyte Proliferation and Apoptosis
2
EdU Labeling and Quantification in Oocytes
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To label the new synthesized DNA, the oocytes were cultured in M2 medium with 10 μM 5-ethynyl-2’-deoxyuridine (EdU) (beyotime, ST067). Then oocytes were fixed with 4% Paraformaldehyde for 15 minutes and permeated with 0.3% Triton X-100 for 15 minutes. After incubation with primary and secondary antibodies, oocytes were treated with the click reaction buffer (beyotime, C0071S) at RT for 1 hour. Then oocytes were washed with PBST for 5 times and stained with Hoechst before observation. To compare the EdU signal sizes, we measured the max length of EdU signals with the Fiji software.
3
Proliferation Tracking in Enteric Glia
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Seven days after administration of RepSox (3 mg/kg), GFAP-Cre:Rosa26-tdTomato mice were given an intraperitoneal injection of EdU (Beyotime, ST067, 50 mg·kg−1·d−1). After 14 days of RepSox administration, the mice were sacrificed, the LMMP tissue was stripped and fixed with 4% PFA at 4 °C for 2 h, and then, the tissue cells were stained for EdU according to the procedures of the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, C0071S). For co-staining with HuCD, the LMMP tissue was incubated with HuCD primary antibody overnight and labelled with the corresponding secondary antibody.
4
In Vivo and In Vitro Cell Proliferation Labeling
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For in vivo labeling of cell proliferation, EdU (Beyotime, ST067) was dissolved in PBS and intraperitoneally injected at a dose of 50 mg/kg 24 h prior to the experimental analysis. For in vivo cell differentiation labeling, EdU was injected at a dose of 200 mg/kg for 3 days and analyzed 30 days later. For in vitro cell proliferation labeling, EdU was added to the NSCs suspension at a final concentration of 10 μM and incubated for 4 h before analysis. To monitor the penetration of EdU, brain frozen slices or cell staining were performed according to the manufacturer's instructions (Beyotime, C0075S). Briefly, the corresponding working solution was prepared and incubated at 37 °C for 40 min, followed by immunostaining analysis.
5
EdU Labeling for Cell Proliferation Analysis
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The cells were seeded in a 24-well plate and added with EdU (ST067, Beyotime, Shanghai, China) at 10 µmol/L concentration. After incubation for 2 h, cells (6–10 fields) were randomly observed under a fluorescence microscope (FM-600, Shanghai Pudan Optical Instrument Co., Ltd.). The number of EdU-positive cells in each field was counted [53 (link)]. EdU labeling rate (%) was calculated as the number of positive cells/(number of positive cells + number of harmful cells) × 100%.
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