Jul 22, 2020
Autofluorescence Reduction and Imaging Marmoset NHP Tissue with a TissueFAXS Imaging System
This protocol is a draft, published without a DOI.
- 1Massachusetts Institute of Technology
Protocol Citation: Guoping Feng Lab 2020. Autofluorescence Reduction and Imaging Marmoset NHP Tissue with a TissueFAXS Imaging System . protocols.io https://protocols.io/view/autofluorescence-reduction-and-imaging-marmoset-nh-biunkeve
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: July 21, 2020
Last Modified: July 22, 2020
Protocol Integer ID: 39534
Autofluorescence Reduction
Autofluorescence Reduction
10m
10m
1) Wash samples in 1X PBS for 00:02:00 .
2) Stain samples free floating with .05 % volume TrueBlack in 70 % volume Ethanol for 00:03:30
3) Rinse samples in 1X PBS for 00:10:00 .
4) Mount samples on a slide with ProLong™ Gold Antifade Mountan.
Imaging Samples
Imaging Samples
1w
1w
Image samples with a 5x overview scan to determine the tissue regions and boarders.
Switch to 20x and image both slices with DAPI and 488 to determine the location of GFP+ cells.
Create "Defined Regions" around GFP+ cells.
Once these defined regions are created, switch to the 40x water immersion objective and "Force Focus" on multiple points across the tissue to find upper and lower bounds for focusing.
Compute focus points for each defined region -- Options, Focus (1x1) -- and set your upper and lower bounds for imaging.
Image each defined region with 0.5 µm steps so that cells can be reconstructed for morphology.
Export the FOVs for morphology reconstruction. Be sure to uncheck “Use Stich” as the stitching is done later in the reconstruction pipline. Save the Z-stack with "Original" and "16-bit" format.