Hello,
I am a first year PhD student and I am working on Cerebral amyloid angiopathy (CAA). I would like to investigate the presence of my proteins of interest in a CAA/Alzheimer's disease mouse model, but I am unsure about how to section their brains in order to better visualise the vessels. Would you recommend coronal or sagittal sections? And why?
Thanks.
Kind regards,
Mark
If you want to specifically visualize vessels the coronal or sagittal sections are equally effective, however, you are encouraged to have thicker slices of the brain i.e. 20-50 um so you can have more vessel length in the slice (you will need to perform confocal z-stacking to see the vessels properly within the thick slice). In terms of detecting the vessels, you can perfuse the mouse intracardially with FITC-Dextran that will visualize the vasculature and provide insight on any leaky vessels (you will see the leakage of the dextran into the brain tissue). If you use the dextran you will need to use cryosectioning as paraffin embedding may damage the FITC. You can also use CD31 staining or CD31 staining combined with ZO-1 that will also show endothelial cells and vessel wall integrity.
Hi Anton Lennikov
Many thanks for the detailed and specific insight!
Dear Marco!
You can also look at this protocol:
Immunohistochemical and histochemical studies
To prepare brain sections, mice were anesthetized with medetomidine–midazolam–butorphanol and then transcardially perfused with PBS first and with 0.1 M phosphate-buffered 4% paraformaldehyde. Brains were removed and sequentially immersed in the same fixative for 16 h and then phosphate-buffered 30% sucrose for 16 h at 4°C. Sagittal or coronal (30-μm thickness) sections were immunostained using the floating method. Briefly, sections were permeabilized with 0.3% Triton X-100 in 1% BSA-PBS for 30 min first and with 5% goat serum-PBS for 60 min. The sections were then incubated with primary and secondary antibodies and DAPI. Detailed descriptions of the primary and secondary antibodies used are provided in Supplementary Table 1. For the detection of amyloidosis, 1-fluoro-2,5-bis(3-carboxy-4-hydroxystyryl)benzene (FSB) was used21. Stained sections were observed using an Olympus FV-1000 confocal microscope. We quantified immunoreactive areas using MetaMorph imaging software (Universal Imaging Corp., West Chester, USA) as previously described42. To reduce the variance among tissue sections, data were averaged from at least four sections per mouse.
https://www.biorxiv.org/content/10.1101/2020.07.22.215418v1.full
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