Visualization of Axonal Projection Patterns of Embryonic Motor Neurons in Drosophila

Samenvatting

Source: Jeong, S. Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila. J. Vis. Exp. (2017)

This video demonstrates the visualization of axonal projections in Drosophila embryos. Immunolabeling of motor neurons, precise dissection to expose neuronal structures, and differential interference contrast microscopy collectively reveal detailed neuronal architecture, emphasizing the axonal projection patterns.

Protocol

1. Preparation

1. Prepare 500 mL of phosphate-buffered saline (PBS) with t-Octylphenoxypolyethoxyethanol (PBT) solution by adding 0.5 g of bovine serum albumin (BSA) and 0.5 mL of t-Octylphenoxypolyethoxyethanol (see the Table of Materials) to 500 mL of 1X PBS and stirring for at least 30 min. Store at 4 °C. Use when relatively fresh and store the solution in a clean bottle.
2. Make 10 mL of 4% paraformaldehyde by adding 2.5 mL of 16% stock paraformaldehyde solution and 1 mL of 10x PBS to 6.5 mL of deionized water. Store at 4 °C and use within one week.
   CAUTION: Avoid skin contact with paraformaldehyde solution because it is hi....

Resultaten

Figure 1: Staging of embryos based on the projection patterns of ISN and ISNb motor axons. (A).......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Bovine serum albumin	Sigma-Aldrich	A7906
Triton X-100	Sigma-Aldrich	X100	t-Octylphenoxypolyethoxyethanol
16% Paraformaldehyde solution	Ted Pella	18505
Sodium chloride	Sigma-Aldrich	S5886
Potassium chloride	Sigma-Aldrich	P5405
Sodium phosphate dibasic	Sigma-Aldrich	30435
Sodium phosphate monobasic	Sigma-Aldrich	71500
X-Gal Substrate	US Biological	X1000	X-Gal (5-Bromo-4-chloro-3-indolyl-b-D-galactoside galactopyranoside)
Dimethyl sulfoxide	Sigma-Aldrich	D4540
Magnesium chloride	Sigma-Aldrich	M8266
Potassium hexacyanoferrate(II) trihydrate	Sigma-Aldrich	P9387
Potassium hexacyanoferrate(III)	Sigma-Aldrich	244023
Hydrogen peroxide	Sigma-Aldrich	216763
3,3'-diaminobenzidine tetrahydrochloride	Sigma-Aldrich	D5905
Agar	US Biological	A0930
Sucrose	Fisher Scientific	S5-3
Tegosept (Methy 4-Hydroxybenzoate)	Sigma-Aldrich	H5501
Culture dish (60 mm)	Corning	430166
Tricon beaker	Simport	B700-100	This is used to make a plastic beaker cage for embryo collection.
Yeast	Societe Industrielle Lesaffre	Saf Instant Yeast Red
Cotton swab (Wooden single tip cotton PK100)	VWR	14220-263
Eppendorf tube (1.5 ml)	Sarstedt	#72.690
Bleach	The Clorox Company	Clorox
Heptane	Sigma-Aldrich	246654
Methanol	J.T. Baker	UN1230
Normal goat serum	Life Technologies	16210-064
Anti-FasciculinII antibody	Developmental Studies Hybridoma Bank	1D4 anti-Fasciclin II
Goat anti-mouse-HRP antibody	Jackson Immunoresearch	115-006-068	AffiniPure F(ab')2 fragment goat anti-mouse IgG+IgM (H+L)(min X Hu, Bov, Hrs Sr Prot
Glycerol	Sigma-Aldrich	G9012
Slide glass	Duran Group	235501403
Coverslip	Duran Group	235503104	18 x 18 mm
1 ml Syringe	Becton Dickinson Medical(s)	301321
Tungsten needle	Ted Pella	#27-11	Tungsten Wire, ø0.13mm/6.1m (ø.005"/20 ft.)
Nutator (Mini twister)	Korean Science	KO.VS-96TWS	Alternatively, BD clay adams brand nutator (BD 421125)