High-Resolution Three-Dimensional Confocal Imaging of the Blood-Brain Barrier in a Mouse Brain Section

Samenvatting

Source: Villaseñor, R and Collin. L. High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis. J. Vis. Exp. (2017)

This video demonstrates high-resolution 3D imaging of the blood-brain barrier (BBB) using confocal microscopy, showing antibody-stained mouse brain sections to analyze BBB components and intracellular structures, applicable for studying brain homeostasis, neurovascular interactions, and disease-related BBB dysfunction.

Protocol

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

1. High-resolution Confocal Imaging of the Blood-brain Barrier
2. Perform image acquisition with a suitable laser-scanning confocal microscope (see Table of Materials) with laser lines at 405, 488, 561, and 633 nm for excitation of fluorophores in the blue, green, orange, and red regions of the light spectrum. For image acquisition, use a 63X oil objective with a numerical aperture of 1.4.
3. In the acquisition menu of the software that controls the microscope, set the image acquisition parameters. In the drop-down menu of ‘Im....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Rat monoclonal antibody clone ER-MP12 against CD31/PECAM	Novus Biologicals	MCA2388	Labels Brain Endothelial CellsUse at dilution 1:100detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400
Rat monoclonal antibody against Podocalyxin	R&D Systems	MAB1556	Labels Lumen of capillariesUse at dilution 1:100detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400
Rabbit polyclonal antibody against CollagenIV	Biotrend	BT21-5014-70	Labels Basement membrane of capillariesUse at dilution 1:100detect with donkey polyclonal anti-rabbit IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400
Goat polyclonal antibody against CD13	R&D Systems	AF2335	Labels pericytesUse at dilution 1:100detect with donkey polyclonal anti-goat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400
Mouse monoclonal antibody clone G-A-5 against GFAP coupled to Cy3	Abcam	ab49874	Labels AstrocytesUse at dilution 1:100
Rabbit polyclonal antibody against Iba-1	Wako	019-19741	Labels MicrogliaUse at dilution 1:100detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400
Rat monoclonal antibody ABL93 gainst LAMP2	Fitzgerald	10R-CD107BBMSP	Labels LysosomesUse at dilution 1:100detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400
Donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor594	LifeTechnologies	A21203	Use at dilution 1:400
Donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor488	LifeTechnologies	A21202	Use at dilution 1:400
Goat polyclonal antibody against mouse IgG (H+L) AlexaFluor555	LifeTechnologies	A21422	Use at dilution 1:400
Fluorescent Mounting medium	Dako	S3023	Dako fluorescent mounting medium
Laser Scanning Confocal microscope	Leica Microsystems	NA	Leica TCS SP8 X with HyD detectorsand White light laser
Image processing software	Leica Microsystems	NA	Leica Application Suite AF version 3.1.0 build 8587
Image analysis software	Bitplane scientific software	NA	Imaris version 7.6.5 build 31770 for x64
Fluorescence SpectraViewer	ThermoFisher Scientific	NA	https://www.thermofisher.com/ch/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html