Fluorescence Immunostaining of Rat Hippocampal Neurons within a Microfluidic Chip

Abstract

Source: Nagendran, T., et al., Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons. J. Vis. Exp. (2018)

This video demonstrates immunofluorescence staining of rat hippocampal neurons cultured within a microfluidic chip to analyze neuronal maturation and connectivity.

Protocol

1. Fluorescence Immunostaining within the Chip

1. Prepare 4% formaldehyde fixation solution in PBS (4% formaldehyde, 1 µM MgCl₂, 0.1 µm CaCl₂, 120 mM sucrose)
2. Remove most of the media in the wells of the chip (do not dry interior compartments).
3. Immediately add 100 µL of fixation solution to the top wells of the axonal and somatic compartments.
4. After 1 min, add 100 µL of fixation solution to the bottom wells. Fix for 30 min at room temperature.
5. Remove most of the solution from the wells of the chip (do not dry interior compartments). Immediately add 150 µL of PBS to each of ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
XonaChip	Xona Microfluidics, LLC	XC150	150 µm length microgroove barrier
XonaChip	Xona Microfluidics, LLC	XC450	450 µm length microgroove barrier
XonaChip	Xona Microfluidics, LLC	XC900	900 µm length microgroove barrier
XC pre-coat	Xona Microfluidics, LLC	XC Pre-Coat	included with XonaChips
XonaPDL	Xona Microfluidics, LLC	XonaPDL
Neurobasal medium	ThermoFisher Scientific	21103049
Glass Pasteur pipettes	Sigma-Aldrich	CLS7095D5X SIGMA
Pierce 16% formaldehyde	ThermoFisher Scientific	28906
PBS (10x)	ThermoFisher Scientific	QVC0508
Normal goat serum	ThermoFisher Scientific	16210064
Triton X-100	ThermoFisher Scientific	28314
Anti-vGlut1 antibody	NeuroMab	75-066	clone N28/9, 1:100
Alexa Fluor secondary antibodies	ThermoFisher Scientific		17:40
Spinning disk confocal imaging system	Andor Technology	CSU-X1, iXon X3 EMCCD	60x silicone oil & 20x objectives