Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

Summary

This study presents a simplified protocol for tissue processing involving decapitation, fixation, cryosectioning, fluorescence staining, immunostaining, and imaging, which can be extended to confocal and multiphoton imaging. The method maintains efficacy comparable to complex dissections, bypassing the need for advanced motor skills. Quantitative image analysis provides extensive investigative potential.

Abstract

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression patterns. Traditional methods often involve challenges such as performing complex dissection, maintaining tissue integrity, and visualizing specific expression patterns during high-resolution imaging. We present an optimized protocol that combines cryosectioning with fluorescence staining and immunostaining. This method improves tissue preservation and signal clarity and reduces the need for laborious dissection for Drosophila brain imaging. The method entails rapid dissection, optimal fixation, cryoprotection, and cryosectioning, followed by fluorescent staining and immunostaining. The protocol significantly reduces tissue damage, enhances antibody penetration, and yields sharp, well-defined images. We demonstrate the effectiveness of this approach by visualizing specific neural populations and synaptic proteins with high fidelity. This versatile method allows for the analysis of various protein markers in the adult brain across multiple z-planes and can be adapted for other tissues and model organisms. The protocol provides a reliable and efficient tool for researchers conducting high-quality immunohistochemistry in Drosophila neurobiology studies. This method's detailed visualization facilitates comprehensive analysis of neuroanatomy, pathology, and protein localization, making it particularly valuable for neuroscience research.

Introduction

Complex behaviors ranging from social interactions¹, sensory perception and processing², learning³, to movement⁴ are driven by the brain. Neurological disorders are also increasingly common and predicted to increase with time^5,6. It is critical to study how the brain works in both health and disease. The central dogma of molecular biology suggests that one of the most important functions of biological units is proteins⁷, and both how much and where they are expressed are critical to understan....

Protocol

1. Preparation of equipment

1. Ensure that the cryostat is powered on and set to -20 °C. Power on the slide warmer or a small incubator, ensuring it is set to 37 °C.
   NOTE: At this stage, labeled slides can be placed on the warmer or incubator and left indefinitely until sectioning.

2. Preparation of solutions

1. Prepare 50 mL of 1x Phosphate buffered saline (PBS), pH 7.4, from 10x PBS stock. Prepare a solution of 4% Paraformaldehyde in PBS with a final volume of 10 mL.
2. Prepare a cleaning solution of 70% ethanol in water in a spray bottle. Prepare a blockin....

Discussion

Here, we present a protocol for precise fluorescent imaging of cryosectioned Drosophila heads. This is a straightforward approach that has several important positives. Namely, the methods are simple enough that anyone with basic laboratory safety training could complete, they are adaptable to measure the expression of any protein that high-quality antibodies exist for, and they allow for precise measurement of both how much a protein of interest is expressed and where that expression occurs throughout the head. .......

Materials

Name	Company	Catalog Number	Comments
1000 uL Pipette	Eppendorf	3123000063
1000 uL Pipette Tips	Olympus Plastics	23-165R
10X Phosphate Buffered Saline (PBS)	Fisher	J62036.K7	ph=7.4
200 Proof Ethanol	Decon Laboratories	64-17-5
20X Tris Buffered Saline	Thermo Scientific	J60877.K2	pH=7.4
AF750 Goat Anti-Mouse Secondary Antibody	Alexa Fluor	A21037
Anti-Roll Glass	IMEB	AR-14047742497
ApoE Mouse Primary Antibody	Santa Cruz	SC13521
Bovine Serum Albumin	Fisher	9048-46-8
Centrifuge Tubes 1.5 mL	Fisher	05-408-129
Charged Slides	Globe Scientific	1415-15
Cryosectioning Molds	Fisher	2363553
Cryostat	Leica	CM 3050 S
Cryostat Blades	C.L. Sturkey	DT554N50
Distilled Water
Dry Ice	???	???
Fine Forceps	Fine Science Tools	11254-20
Fly Pad	Tritech Research	MINJ-DROS-FP
Hardening mounting Media with Dapi	Vectashield	H-1800
Kimwipes	Kimtech	34120
Microscope	Olympus	SZ61
Nile Red	Sigma	N3013
Optimal Cutting Temperature Compound	Fisher	4585
Orbital Shaker	OHAUS	SHLD0415DG
Paraformaldehyde 20%	Electron Microscopy Sciences	15713
Razor Blades	Gravey	#40475
Spring Scissors	Fine Science Tools	15000-10
Sucrose	Fisher	S5-500