Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Summary

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Abstract

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.

Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.

This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Introduction

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca²⁺ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.^1-3 The original discoveries of embryonic Ca²⁺ signaling were dependent on the use of natural and engineered Ca²⁺ indicators, such as aequorin⁴ and fura-2.⁵ Even with current technology, the detection of transient elevations of Ca²⁺ requires cumbersome analytical tools and does not reveal the targets of such Ca²⁺ signals.....

Protocol

The zebrafish procedures in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University.

1. Preparation of Reagents

1. 4% PFA/PBS. Weigh 8 g of paraformaldehyde (PFA) in the fume hood. While still in the fume hood, dissolve the dry PFA in ~80 ml distilled H₂O with stirring and heating to 50 °C. While stirring, add 3 - 10 drops of fresh 1N NaOH until PFA is completely dissolved and solution clarifies. Remove from heat and add 100 ml 2x phosphate buffered saline (PBS). Bring volume to 200 ml with distilled H₂O. Cool to RT and....

Results

Optimal Conditions for Visualizing Phospho-epitopes

Methods describing the immunolocalization of protein epitopes in zebrafish embryos have been relatively sparse compared to those for localizing mRNAs via in situ hybridization. Fixatives used in localizing protein epitopes in ciliated cells of zebrafish embryos have included 4% PFA/PBS and Dent's fixative, which is a mixture of methanol and DMSO.^1.......

Discussion

The PFA/methanol method was developed in this laboratory with the primary objective of optimizing the immunolocalization of the phospho-T²⁸⁷-CaMK-II epitope during zebrafish development. This method successfully localized P-CaMK-II during the formation of several ciliated organs including the zebrafish KV,¹² inner ear¹⁴ and kidney.¹³ Particularly at the KV stage, this technique was necessary. The success of this method is likely due to a combination of a) minimization of autofl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-phenyl-2-thiourea (PTU)	Sigma	P-7629	0.12% Stock solution. Dilute 1:40 in system water
Alexa488 anti-mouse IgG	Life Technologies	A11001	Goat polyclonal, use at 1:500
Alexa488 anti-rabbit IgG	Life Technologies	A11008	Goat polyclonal, use at 1:500
Alexa488 phalloidin	Life Technologies	A12379	Preferentially binds to F-actin
Alexa568 anti-mouse IgG	Life Technologies	A11004	Goat polyclonal, use at 1:500
Alexa568 anti-rabbit IgG	Life Technologies	A11011	Goat polyclonal, use at 1:500
anti-acetylated α-tubulin	Sigma	T7451	Mouse monoclonal, use at 1:500
anti-phospho-T287 CaMK-II	EMD Millipore	06-881	Rabbit polyclonal, use at 1:20
anti-total CaMK-II	BD Biosciences	611292	Mouse monoclonal, use at 1:20
Ethanol	Fisher	S96857	Lab grade, 95% denatured
Forceps	Fine Science Tools	11252-20	Dumont #5
Glass coverslips	VWR	16004-330	#1  thickness
Glass microscope slides	Fisher	12-550-15	Standard glass slides
Methanol	Fisher	A411	Store in freezer
Microcentrifuge tubes	VWR	20170-038	capped tubes, not sterile
Normal goat serum	Life Technologies	16210-064	Aliquot 1 ml tubes, store in freezer
Paraformaldehyde	Sigma	P-6148	Reagent grade, crystalline
Phosphate buffered saline (PBS)	Quality Biological	119-069-131	10x stock solution or made in lab
Triton X-100	Sigma	BP-151	10% solution in water, store at RT
Tween-20	Life Technologies	85113	10% solution in water, store at RT
Compound microscope	Nikon	E-600	Mount on vibration-free table
C1 Plus two-laser scanning confocal	Nikon	C1 Plus	Run by EZ-C1 program, but upgrades use "Elements"