Immunostaining of Fluorescently Labeled Astrocytes in a Mouse Brain Tissue Section

Abstract

Source: Eaker, A. R., et al. Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections. J. Vis. Exp. (2022)

This video demonstrates the procedure for immunostaining a fixed mouse brain tissue section with fluorescently labeled astrocytes using primary and secondary antibodies and capture the images of the stained astrocytes through confocal microscopy.

Protocol

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

1. Immunostaining

NOTE: Perform all washes and incubations on an orbital platform shaker set to approximately 100 rpm. All steps are performed at room temperature, except the primary antibody incubation, which is performed at 4 °C. Prepare mounting media ahead of time by combining the ingredients in a 50 mL tube and mixing on a nutator overnight. Protect from light and store at 4 °C for up to 2 months. If the endogenous fluorescent signal is sufficie....

Results

Figure 1: Overview of workflow for analyzing astrocyte territory volume. (A) The protocol requires a mouse with sparse or mosaic fluorescent labeling of astrocytes. (B) The mouse is perfused, and then the brain is resect.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x TBS (tris-buffered saline)			30 g Tris, 80 g NaCl, 2 g KCl, HCl to pH 7.4, dH2O to 1 L; store at room temperature (RT)
12-well plate	Genesee Scientific	25-106MP
1x TBS			100 mL 10x TBS + 900 mL dH2O; store at RT
24-well plate	Genesee Scientific	25-107MP
CD1 mice	Charles River	22
Confocal microscope	Olympus	FV3000RS
Coverslips	Fisher Scientific	12544E
DAPI	Invitrogen	D1306
GFP antibody	Aves Labs	GFP1010
Glycerol	Thermo Scientific	158920010
Goat anti-chicken 488	Invitrogen	A-11039
Goat anti-rabbit 594	Invitrogen	A11037
Goat Serum	Gibco	16210064
Imaris	Bitplane	N/A	Version 9.8.0
MATLAB	MathWorks	N/A
Nailpolish	VWR	100491-940
O.C.T.	Fisher Scientific	23-730-571
Oil	Olympus	IMMOIL-F30CC	Specific to microscope/objective
Operating Scissors 6"	Roboz	RS-6820
Orbital platform shaker	Fisher Scientific	88861043	Minimum speed needed: 25 rpm
Paintbrush	Bogrinuo	N/A	From Amazon, "Detail Paint Brushes - Miniature Brushes"
Paraformaldehyde	Sigma-Aldrich	P6148
Pasteur pipet (5.75")	VWR	14672-608
Pasteur pipet (9")	VWR	14672-380
Potassium chloride	Sigma-Aldrich	P9541-500G
RFP antibody	Rockland	600-401-379
Slides	VWR	48311-703
Sodium chrloide	Fisher Scientific	BP358-212
Sodium hydroxide	Sigma-Aldrich	S5881
Sucrose	Sigma-Aldrich	S0389
Transfer Pipet	VWR	414004-002
Tri-bromoethanol	Sigma-Aldrich	T48402
Tris(hydroxymethyl)aminomethane	Thermo Scientific	424570025
Triton X-100	Sigma-Aldrich	93443
Triton X-100 (high-quality)	Fisher Scientific	50-489-120