Abstract
In this protocol, we describe a method to visualize and map dural lymphatic vessels in-vivo using magnetic resonance imaging (MRI) and ex-vivo using histopathological techniques. While MRI protocols for routine imaging of meningeal lymphatics include contrast-enhanced T2-FLAIR and T1-weighted black-blood imaging, a more specific 3D mapping of the lymphatic system can be obtained by administering two distinct gadolinium-based MRI contrast agents on different days (gadofosveset and gadobutrol) and subsequently processing images acquired before and after administration of each type of contrast. In addition, we introduce methods for optimal immunostaining of lymphatic and blood vessel markers in human dura mater ex-vivo.
Highlights
[Background] Among the causes of immune privilege in the brain is the absence of parenchymal lymphatic vessels
We describe a way to: (1) visualize the lymphatic vessels in-vivo in the dura mater using magnetic resonance imaging (MRI) of the head, and (2) assess the local presence of lymphatic vessels using optimized immunostaining methods (Absinta et al, 2017)
If samples vary in disease type or treatment status, simple comparative statistics may be computed on the count and diameter data (Figure 5)
Summary
Tris buffered saline 10x, pH 7.4 (KD Medical, catalog number: RGF-3385) 13. CD31 antibody (Abcam, catalog number: ab28364) 18. CD68 (KP-1) antibody (Thermo Fisher Scientific, Invitrogen, catalog number: MA5-13324) 22. CD3 antibody (Agilent Technologies, Dako, catalog number: A0452) 23. Antibody Diluent (Agilent Technologies, Dako, catalog number: S0809) 24. PV Poly-HRP Anti-Mouse IgG (Leica Biosystems, catalog number: PV6114) 25. PV Poly-HRP Anti-Rabbit IgG (Leica Biosystems, catalog number: PV6119) 26. ImmPRESSTM-AP Anti-Rabbit IgG (Vector Laboratories, catalog number: MP-5401) 27. ImmPRESSTM-AP Anti-Mouse IgG (Vector Laboratories, catalog number: MP-5402) 28. Blue buffer 8 (Leica Biosystems, catalog number: 3802916) 34. Tween 20 (Agilent Technologies, Dako, catalog number: S1966) 37.
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