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

Abstract

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 ‘Image size’, select a value of 1024 x 1024 pixels. Modify the pixel size to a value between 200 and 300 nm by adjusting the value of the ‘Zoom’ menu.”
   NOTE: For the analysis of intracellular structures, reduce the pixel size to 75 nm. This image size setting substantially increases the acquisition time and can be reduced to 512 x 512 pixels to decrease the acquisition time by imaging a smaller field of view.
4. In the 'Acquisition Speed' drop-down menu, select a value of 400 Hz, i.e. 400 lines per second. In the 'systems settings' menu, select the 'Pixel depth' drop down menu, and change the value to 12 bit.
5. In the microscope software, within the 'acquisition' tab, toggle on the option for sequential frame acquisition. Set the optical section thickness to a value between 0.75 and 1 µm for each channel by modifying the value in the 'Pinhole' menu.
6. In the panel for fluorescent excitation, activate the laser required to optimally excite the fluorophores in the sample, for example, a 488 nm laser line for a green-emitting fluorophore.
   NOTE: Use a fluorophore spectra viewer (see Table of Materials) to select the adequate excitation laser.
7. In the panel for fluorescent detection, move the slider to select the wavelengths that will be measured in each channel, for example, between 510 and 550 nm for a green-emitting fluorophore.
   NOTE: Use a fluorophore spectra viewer (see Table of Materials) to select the adequate detection wavelengths.
8. Add a drop of immersion oil with a refractive index of 1.52 on top of the coverslip with the brain section to match the refractive index of the glass coverslip and objective. Place the sample in the microscope and turn on the epifluorescent lamp to visualize the sample using the microscope binoculars.
9. Using the buttons on the microscope stand, change the filter wheel to select a filter appropriate for visualizing DAPI (4',6-diamidino-2-phenylindole)-stained nuclei. Use the coarse focus to bring the signal from DAPI-stained nuclei into focus.
10. Using the buttons on the microscope stand, change filters to visualize the signal from vascular markers (for example, CollagenIV or CD31) and center the field of view on an individual capillary segment.
11. Press the button to start the live-scan mode. During scanning, adjust the gain and laser intensity for each channel to maximize the dynamic range of the images and avoid pixel saturation.
    NOTE: Use a look-up table which labels saturated pixels to visually assess over-saturation. To avoid signal over-saturation, adjust the settings with the sample which is expected to have the highest fluorescent signal.
12. Set the value for line averaging to 2.
    NOTE: When using higher acquisition speeds, increase this value to reduce noise.
13. Establish begin and end sections for performing optical z-stacks that span the whole volume of the capillary. Use a step size of 0.45 µm.
    NOTE: If images will be used for subsequent quantification, keep the same acquisition settings for the complete data set.
14. For quantification, acquire 10 to 20 z-stacks per section from at least three different mice for statistical comparisons.
    NOTE: Acquire the full data set in the same session to reduce the variability arising from sample bleaching and laser intensity fluctuations.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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