Useful tip for better zebrafish whole-mount IHC staining

The zebrafish (Danio rerio) is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. Interestingly, this convenient model is compatible with large-scale genetic analysis for developmental & regenerative biology, oncology, environment & toxicology, neurobiology…

Analysis of protein expression and localization by zebrafish immunostaining (IHC and IF), in sections or whole-mount preparations (IHC-Wm), is easy to perform with zebrafish research primary antibodies (such as the Elite Antibodies from Genetex). However, to further sharpen the signal-to-noise ratio for each antibody requires tedious protocol optimization.

Nevertheless, there is a very simple tip to optimize IHC-Wm results. This simple (but efficient) optimization is based on “a heating step”, in accordance with a report by Inoue D. and Wittbrodt J. for fluorescent immunostaining.

Follow this tip for optimized Zebrafish Immunostaining

  1. Fixation & rehydration
  2. Heat samples in Tris-HCl buffer (150mM, pH9.0) at 70o°C for 15min
  3. Permeabilization & subsequent immunostaining

This optimization is incorporated into Genetex’s standard zebrafish protocol. This heating step significantly enhances signal strength via efficient antigen retrieval and is widely applicable to conventional zebrafish immunostaining.

 See it in action!

How? By following Genetex’s protocol for whole-mount preparation immunostaining with 5 selected Zebrafish Antibodies guaranteed by Genetex and tebu-bio!

Pax2a antibody (GTX128127)

GTX128127 anti -Pax2a Genetex tebu-bio IHC for Zebrafish

Pax2a protein detected at Pronephric duct and neurons on whole-mount paraformaldehyde-fixed zebrafish embryos by IHC. Pax2a antibody (GTX128127) was 1:200 diluted.

anti Pax2a for Zebrafish IHC GTX128127 Genetex tebu-bio

Pax2a detection (GTX128127) in zebrafish whole-mount.

myl7 antibody (GTX128346 )

 

Zebrafish (Danio rerio) heart Genetex tebu-bio

Myl7 antibody (GTX128346; dilution 1/100) detects Myl7 protein in the heart of whole-mount paraformaldehyde-fixed zebrafish embryos by immunohistochemical analysis.

 

alpha cardiac actin antibody (GTX124462)

alpha cardiac actin antibody GTX124462 in IHC-Wm on Zebrafish

Immunohistochemical analysis (whole mount) of Zebrafish embryo, using actc1a (GTX124462) antibody (1/200 dilution).

GFP antibody (GTX 113617)

GTX113617 in IHC-Wm by Genetex tebu-bio

Immunohistochemical analysis (whole mount) of transgenic zebrafish embryo, using GFP(GTX113617) antibody at 1/200 dilution.

vmhc anti (GTX127331)

GTX127331 IHC-wm Zebrafish vmhc staining

Immunohistochemical analysis of Zebrafish whole-mount embryos, 24 hpf, using Ventricular Myosin Heavy Chain (vmhc; GTX127331) antibody (1/100 dilution).

What about you?

Which tips or tricks would you recommend to fellow researchers also working on the zebrafish model?

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