Immunofluorescence Assay to Detect PM2.5-Mediated DNA Damage in Fish Embryos
Published: April 30, 2023
Abstract
Source: Huang, Y., et al. Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts. J. Vis. Exp. (2021).
This video demonstrates a highly sensitive immunofluorescence-based assay for detecting PM2.5-induced oxidative damage in the hearts of zebrafish embryos by measuring the levels of 8-OHdG and γH2AX, which indicate DNA damage and double-strand DNA breaks, respectively.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. PM2.5 sampling and organic compound extraction NOTE: PM2.5 was collected in an urban area in Suzhou, China, August 1-7, 2015, as described previously. Bake 47 mm quartz membrane filters in a 500 °C muffle furnace for 2 h to remove the organic components. P…
Disclosures
The authors have nothing to disclose.
Materials
| 8-OHdG Antibody | Santa Cruz Biotechnology, USA | sc-66036 | Primary antibody |
| Analytical balance | Sartorius,China | BSA124S | |
| BSA | Solarbio,Beijing,China | SW3015 | For blocking |
| DAPI | Abcam, USA | ab104139 | For nuclear counterstain. |
| DMSO | Solarbio,Beijing,China | D8371 | |
| Fluorescence microscope | Olympus, Japan | IX73 | For imaging fluorescence signals |
| Goat Anti-Rabbit IgG Cy3 | Carlsbad,USA | CW0159 | Secondary antibody |
| Goat Anti-Rabbit IgG FITC | Carlsbad,USA | RS0003 | Secondary antibody |
| N-Acetyl-L-cysteine(NAC) | Adamas-Beta, Shanghai, China | 616-91-1 | |
| Orbital shaker | QILINBEIER,China | TS-1 | |
| Paraformaldehyde | Sigma,China | P6148 | Make 4% paraformaldehyde for fixation. |
| Phosphate Buffered Saline | HyClone,USA | SH30256.01 | Prepare 0.1% Tween in PBS for washing. |
| PM2.5 sampler | TianHong,Wuhan, China | TH-150C | For 24-hr uninterrupted PM2.5 sampling. |
| Re-circulating aquaculture system | HaiSheng,Shanghai,China | The zebrafish was maintained in it. | |
| Soxhlet extractor | ZhengQiao,Shanghai, China | BSXT-02 | For organic components extraction. |
| Stereomicroscope | Nikon,Canada | SMZ645 | For heart dissection from zebrafish embryos. |
| Tricaine methanesulfonate (MS222) | Sigma,China | E10521 | To anesthetize zebrafish embryos |
| Tween 20 | Sigma,China | P1379 | |
| γH2AX Antibody | Abcam, USA | ab26350 | Primary antibody |
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Cite This Article
Immunofluorescence Assay to Detect PM2.5-Mediated DNA Damage in Fish Embryos. J. Vis. Exp. (Pending Publication), e21281, doi: (2023).