Immunohistochemical Analysis of Zebrafish Embryos

The embryos were fixed in 4% PFA (PBS) for 1 h at room temperature. After washing in PBST (0.1% Tween 20 in PBS), the tails of embryos were clipped for genomic DNA extraction for genotyping, and the rest parts were mounted with 1.5% agarose dissolved in 30% sucrose PBS and then equilibrated overnight at 4°C. The blocks were mounted with OCT compound (Sakura). The sections were cut serially to an 11-μm thickness and collected on poly-L-lysine coated glass slides (CITOGLAS, 188105). Immunofluorescence staining was performed as described (Guan et al., 2016 (link)). Rabbit polyclonal antibody against zebrafish Fabp10a (1:500) and mouse monoclonal antibody against zebrafish Bhmt (1:500) were generated by Hangzhou HuaAn Biotechnology Company. P-H3 antibody was purchased from Santa Cruz (sc-8656-R, 1:600), PCNA antibody from Sigma (P8825, 1:1000), and 2F11 monoclonal antibody from Abcam (ab71286, 1:500). Alexa Fluor 647-labeled secondary antibody was used for visualization.

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Gao C., Huang W., Gao Y., Lo L.J., Luo L., Huang H., Chen J, & Peng J. (2018). Zebrafish hhex-null mutant develops an intrahepatic intestinal tube due to de-repression of cdx1b and pdx1. Journal of Molecular Cell Biology, 11(6), 448-462.

Publication 2018

P-H3 antibody sc-8656-R ab71286

Agarose Alexa fluor 647 Antibody Bhmt Embryos Genomic Immunofluorescence Lysine Monoclonal antibody Mouse Pcna Poly Rabbit Sucrose Tails Tween 20 Zebrafish

Corresponding Organization : Zhejiang University

Other organizations : Southwest University

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Variable analysis

independent variables

Fixation of embryos in 4% PFA (PBS) for 1 h at room temperature
Clipping of tails of embryos for genomic DNA extraction
Mounting of the rest of the embryos with 1.5% agarose dissolved in 30% sucrose PBS and equilibration overnight at 4°C
Sectioning the blocks with OCT compound to an 11-μm thickness

dependent variables

Immunofluorescence staining of sections using antibodies against Fabp10a, Bhmt, P-H3, and PCNA

control variables

Use of poly-L-lysine coated glass slides for sections
Use of Alexa Fluor 647-labeled secondary antibody for visualization

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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