Fluorescence Immunostaining of Rat Hippocampal Neurons within a Microfluidic Chip

Samenvatting

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 the top wells of the axonal and somatic compartments. Wait 2 min for the PBS to flow into the bottom wells.
6. Repeat step 1.5 twice.
7. Remove most of the PBS from the wells of the chip. Immediately add 150 µL of PBS with 0.25% TritonX-100 to each of the top wells of the axonal and somatic compartments. Wait for 15 min.
8. Remove most of the liquid from the wells of the chip and immediately add 150 µL of blocking solution (10% normal goat serum in PBS) to each of the top wells of the axonal and somatic compartments. Wait for 15 min.
   NOTE: Effective blocking solutions should be specific to the secondary antibody, e.g., for a donkey anti-sheep secondary antibody, use donkey serum in the blocking solution.
9. Remove most of the liquid from the wells of the chip and immediately add 100 µL of primary antibody (or antibodies) in 1% normal goat serum in PBS to each of the top wells of the axonal and somatic compartments. Cover to minimize evaporation and wait for 1 h at room temperature or 4 °C overnight.
10. 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 the top wells of the axonal and somatic compartments. Wait 5 min for the PBS to flow into the bottom wells.
11. Repeat step 1.10 twice.
12. Remove most of the liquid from the chip's wells and immediately add 100 µL of secondary antibody (or antibodies) in PBS to each of the top wells of the axonal and somatic compartments. Cover to minimize evaporation and wait for 1 h at room temperature.
    NOTE: Refer to the manufacturer's instructions for the recommended dilution of secondary antibodies.
13. Repeat steps 1.10-1.11.
14. If imaging within 1 day of immunostaining, keep the chip filled with PBS. If the chip will be stored longer than 1 day before imaging, wrap the dish containing the chip in paraffin film to prevent evaporation and store it at 4 °C until ready to image.
15. For longer-term storage of samples, mounting media (e.g., Fluoromount-G) can be used.
    1. Remove most of the liquid from the wells of the chip. Use a 1 mL disposable plastic pipet to add 2 drops of mounting media to each of the top wells of the axonal and somatic compartments.
    2. Tilt the chip to encourage the flow of the mounting media through the channels. After 5 min, add 2 drops to the bottom wells. Wait for 1 h before imaging.
       NOTE: After using mounting media it will not be possible to re-probe for other targets.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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