Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

Summary

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Abstract

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis¹, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation^2-5 and degeneration⁶.

The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron^7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape^4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring ^12,13.

In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Protocol

1. Preparation of reagents

Notes before beginning: Dissection and immunohistochemical staining are carried out in a magnetic chamber and the larva is pinned down using specially shaped insect pins. Detailed instructions on the construction of a magnetic chamber, and preparation of these pins can be found in related references ^14,15. In brief, a 1x1cm square hole is cut into a magnetic sheet and a coverslip affixed to the rear of the sheet to make a small chamber. The sides of the chamber are sealed with epoxy glue; after this glue has set the chamber is washed several times with 70% ethanol before use. Dissection i....

Discussion

To understand how complex cell shapes are achieved it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to assay microtubule organization of dendritic arborization neuron sensory dendrites. In addition to staining sensory neurons, this method achieves robust immunohistological staining of trachea, muscles and other body wall tissues.

We use this protocol to examine microtubule organization in the developing sensor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps	Dumont	11251-20
Microscissors	Fine Science Tools	15000-08
Mouse anti-α-tubulin (Clone: DM1A)	Sigma-Aldrich	T9026	Dilution 1/1000
Mouse anti-Futsch (Clone: 22C10), supernatant	Developmental Studies Hybridoma Bank	22C10	Dilution 1/1000
Rat anti-CD8 (Clone: 5H10)	Caltag	MCD0800	Dilution 1/1000
Alexa Fluor 488 anti-mouse IgG	Invitrogen	A-11001	Dilution 1/500
Cy3 anti-Rat IgG	Jackson ImmunoResearch	712-166-150	Dilution 1/200