Real-Time Gene Expression Monitoring in Brain Slices via Three-Dimensional Imaging

Abstract

Source: Shimojo, H., et al. Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis. J. Vis. Exp. (2019)

In this video, cortical slice cultures from transgenic mouse embryos are used to monitor gene expression through Three-dimensional imaging. Bioluminescence, fluorescence, and bright-field images are used to visualize real-time gene expression during neuronal differentiation within the native tissue.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Visualization of luciferase reporter expression in slice cultures
   1. Start the luminescence live-imaging system before starting the dissection. Use the following conditions for cortical slice cultures: 40% O₂, 5% CO₂ and 37 °C.
   2. Put the immersion oil on the 40x objective lens. Put the sample dish on the microscope stage. Acquire the fluorescent test image and set the position and focus plane to the region of interest under the illumination of excitation light.
   3. Run the time-lapse acquisition by 3-dimensional (luminescence, fluorescence, and bright field) acquisitions for 24 h (Figure 1F-H). For luminescence image acquisition, use the following camera settings: low-transfer rate (50 kHz), 2x2/4x4 binning, 10 min/5 min exposure time. For the fluorescence and bright field image acquisition, use the following settings: intermediate-transfer rate (1 MHz), 1x1 binning, and 100 ms exposure time.

Results

Figure 1: Making the cortical slices of the embryonic telencephalon and visualizing Dll1 protein dynami...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chilled water circulator (chiller)	Julabo	Model: F12-ED
Cooled CCD camera	Andor Technology	Model: iKon-M 934
Incubator system	TOKAI HIT	Model: INU-ONICS
Inverted microscope	Olympus	Model: IX81
Inverted microscope	Olympus	Model: IX83
LED illumination device	CoolLED	Model: pE1
MetaMorph	MOLECULAR DEVICES	Model: 40000
Mix gas controller	Tokken	Model: TK-MIGM OLO2
Objective lens	Olympus	Model: UPLFLN 40X O