Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Abstract

Source: Quintanilla, L. J., et al. Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells. J. Vis. Exp. (2019)

This video demonstrates the procedure for staining newly proliferated neural stem cells in brain tissue sections using EdU incorporation, followed by immunofluorescence to visualize specific proteins, enhancing the understanding of cell dynamics and proliferation.

Protocol

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

1. Immunohistochemistry

1. Thymidine analog staining​
   1. Place tissue sections in PBS (phosphate buffered saline) and mount 5−8 sections on a positively charged slide, maintaining serial order from anterior to posterior and the same orientation. Let tissue sections dry to completely adhere to slides, and then draw a border using a hydrophobic pen or PAP pen.
   2. Permeabilize sections with permeabilization buffer (0.5% TBS (tris buffered saline) -tri....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-Ethynyl-2'-deoxyuridine (Edu)	Carbosynth	NE08701
Alexa-488 Azide	Thermo Fisher Scientific	A10266
Anti-Chicken Nestin	Aves	NES; RRID: AB_2314882
Anti-Goat DCX	Santa Cruz	Cat# SC_8066; RRID: AB_2088494
Anti-Mouse Tbr2	Thermo Fisher Scientific	14-4875-82; RRID: AB_11042577
Confocal Software (Zen Black)	Zeiss Microscopy	Zen 2.3 SP1 FP1 (black)
Copper (II) Sulfate Pentahydrate	Thermo Fisher Scientific	AC197722500
Coverslip	Denville Scientific	M1100-02
Plus Charged Slide	Denville Scientific	M1021
Phosphate Buffered Solution (PBS)	Thermo Fisher Scientific	10010031
Super PAP Pen 4 mm tip	PolySciences	24230
Tris Buffered Solution (TBS)	Sigma-Aldrich	T5912
Triton X-100	Sigma-Aldrich	93443