Isolating Fluorescently Labeled Neurons from a Murine Brain

Samenvatting

Source: Paul, A., et al., Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq). J. Vis. Exp. (2018)

This video demonstrates a method to isolate fluorescently labeled neurons from a mouse brain.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Manual Sorting of Fluorescently Labeled Mouse Neurons

1. Pull glass microcapillaries (see Table of Materials) to 10–15 µm exit diameter using a capillary puller with the following settings: heat: 508, pull: blank, vel: blank, time: blank.
2. Attach 120–150 cm flexible silicone tubing (~0.8 mm inside diameter) to a 0.2 µm polyvinylidene difluoride (PVDF) membrane syringe filter and a two-way tubing valve using suitable tubing connectors.
3. Prepare 500 mL of chilled....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
DL-AP5	Tocris	Cat# 0105
CNQX	Tocris	Cat# 1045
TTX	Tocris	Cat# 1078
Protease from Streptomyces griseus	Sigma-Aldrich	Cat# P5147
Carbogen	Airgas	Cat# UN3156
Sylgard 184	Sigma-Aldrich	Cat# 761036
Dissection microscope with fluorescence and bright field illumination with DIC optics. (Leica model MZ-16F).	Leica	Model MZ-16F
Glass microcapillary: Borosilicate capillary tubes 500/pk. OD=1 mm, ID=0.58 mm, wall=0.21 mm, Length=150 mm.	Warner instruments	Model GC100-15, Order# 30-0017
Capillary pipette puller	Sutter Instruments Co	P-97
Vibratome	Thermo Microm	HM 650V
Vibratome tissue cooling unit	Thermo Microm	CU 65