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).

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