Immunofluorescence Assay to Detect PM2.5-Mediated DNA Damage in Fish Embryos

Abstract

Source: Huang, Y., et al. Using Immunofluorescence to Detect PM_2.5-induced DNA Damage in Zebrafish Embryo Hearts. J. Vis. Exp. (2021).

This video demonstrates a highly sensitive immunofluorescence-based assay for detecting PM_2.5-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. PM_2.5 sampling and organic compound extraction

NOTE: PM_2.5 was collected in an urban area in Suzhou, China, August 1-7, 2015, as described previously.

1. Bake 47 mm quartz membrane filters in a 500 °C muffle furnace for 2 h to remove the organic components.
2. Place a filter in a PM_2.5 sampler for 24 h of uninterrupted sampling.
3. Remove the filter and dry for 24 h at room temperature.
....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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