Source: Shull, G. et al. Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection. J. Vis. Exp. (2021)
In this video, we demonstrate the robotic microinjection of fluorescently-labeled polysaccharides into pyramidal neurons in organotypic slices of mouse embryonic brain using an autoinjector. The autoinjector is a robotic platform that utilizes images acquired from a microscope for the precise delivery of biomolecules of interest into single cells.
The authors have nothing to disclose.
Chemicals | |||
Agarose, Low Melt | Carl Roth | Cat# 6351.2 | |
Agarose, Wild Range | Sigma-Aldrich | Cat# A2790 | |
Best-CA 221 Glue | Best Klebstoffe GmbH & Co.KG | Cat# CA221-10ml | |
B-27 Supplement | Thermo Fisher Scientific | Cat# 17504044 | |
Distilled Water | |||
DMEM-F12, CO2 independent (w/o Phenol red) | Sigma-Aldrich | Cat# D2906 | |
DMEM-F12, CO2 independent (with Phenol red) | Sigma-Aldrich | Cat# D8900 | |
HEPES-NAOH, pH 7.2, 1M (HEPES buffer) | Carl Roth | Cat# 9105.3 | |
L-Glutamine, 200 mM | Thermo Fisher Sientific | Cat# 25030024 | |
Mowiol 4-88 | Sigma-Aldrich | Cat# 81381 | |
N-2 Supplement | Thermo Fisher Scientific | Cat# 17502048 | |
Neurobasal Medium | Thermo Fisher Scientific | Cat# 21103049 | |
Nuclease-free water | Thermo Fisher Scientific | Cat# AM9937 | |
O2 (40%), CO2 (5%), N2 (55%) Mix, 50 liters | |||
PBS | |||
Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | Cat# 15140122 | |
Rat serum | Charles River Laboratories | ||
Sodium bicarbonate (NaHCO3) | Merck | Cat# 106323 | |
Sodium hydroxide (NaOH) | Merck | Cat# 106482 | |
Tyrode’s salt | Sigma | Cat# T2145-10x1L) | |
Equipment | |||
Borosilicate glass capillaries, 1.2 mm outer diameter x 0.94 mm inner diameter | Sutter Instruments | Cat# BF-120-94-10 | |
Bottle-top filter system, 500 mL | Corning | Cat# 430769 | |
Computer PC | |||
Custom pressure rig | Custom pressure rig | ||
Electronic pressure regulator | Parker Hannifin | Cat# 990-005101-002 | |
Falcon tubes, 15 mL | Corning | Cat# 430791 | |
Falcon tubes, 50 mL | Corning | Cat# 430829 | |
Flaming/ Brown micropipette puller | Sutter Instruments | Cat# P-97 | |
Forceps, Dumont no. 3 | Fine Science Tools | Cat# 11231-30 | |
Forceps, Dumont no. 5 | Fine Science Tools | Cat# 11255-20 | |
Forceps, Dumont no. 55 | Fine Science Tools | Cat# 11252-20 | |
Heating block | Labtech International | Cat # Dri block Digi2 | |
Inverted fluorescence microscope | Zeiss | Cat# Axiovert 200 | |
Light source | Olympus | Cat# Highlight 3100 | |
Manual pressure regulator | McMaster Carr | Cat# 0-60 PSI 41795K3 | |
Microloader Tips | Eppendorf | Cat# 5242956.003 | |
Microcontroller | Arduino | Cat# Arduino Due | |
Microscope camera Hamamatsu Orca Flash 4.0 V3 | |||
Motorized stage XY for microscope | |||
Pasteur pipettes, plastic | |||
Petri dish, 60 x 15 mm | Greiner | Cat# 628102 | |
Petri dish, 35 x 10 mm | Nunc | Cat# 153066 | |
Petri dish, 34 x 14 mm, including Microwell no. 1.5 cover glass | MatTek | Cat# P35G-1.5-14-C | |
Pipette holder | Warner Instruments | Cat# 64-2354 MP-s12u | |
Pipette and tips | |||
Puller filament, 3.0-mm square box filament | Sutter Instrument | Cat# FB330B | |
Slice culture incubation box | MPI-CBG | Cat# custom made | |
Solenoid valve | Cat# LHDA053321H-A | ||
Tabletop centrifuge | Heraeus | Cat# 5431622 | |
Thermometer | |||
Three-axis Manipulator | Sensapex Inc | Cat# tree-axis uMP | |
Vibratome | Leica | Cat# VT1000s | |
Whole-embryo-culture-system incubator | Ikemoto Company | Cat# RKI-10-0310 | |
Waterbath | |||
Software and Algorithms | |||
Arduino | Arduino | ||
Fiji | RRID: SCR_002285 | ||
Python | Python Software foundation | Python 2.7.12 | |
ZEN | RRID: SCR_013672 |