Apr 16, 2020
Lightsheet Tissue Intake - Photodocumentation and Tracking
- 1University of Florida
- Optical Clearing of Tissue
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Protocol Citation: Seth Currlin, Marda Jorgensen 2020. Lightsheet Tissue Intake - Photodocumentation and Tracking. protocols.io https://dx.doi.org/10.17504/protocols.io.bef6jbre
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: Working
We use this protocol and it's working
Created: March 31, 2020
Last Modified: April 16, 2020
Protocol Integer ID: 35038
Keywords: lightsheet microscopy, tissue processing, tissue tracking
Abstract
In order to track tissue position and orientation each sample is photographed when received for lightsheet microscopy (Figure 1, A). Large samples (>1 cm3), such as the spleen in Figure 1, are cut into 2 mm sections (Figure 1, B) to facilitate tissue clearing, staining, and three-dimensional imaging. These tissues are batch processed and withdrawn from the lipid clearing pipeline as needed (Figure 1, C); at this time a novel identifier is assigned to the sample.
Guidelines
Photodocumentation, in our pipeline, occurs following tissue-hydrogel polymerization and multiple washes in PBS. This serves to remove trace fixatives for safer handling.
Materials
MATERIALS
50 ml conical tubes
Forceps (tweezers), 12.5cm, Blunt EndBio Basic Inc.Catalog #FC003.SIZE.1
Razor bladesFisher ScientificCatalog #12-640
Hexagonal Polystyrene Weighing DishesThermo FisherCatalog #02202103
Safety warnings
Wash tissues of any fixatives, or other dangerous chemicals, prior to manipulating for photodocumentation.
Photograph whole tissue; capture from various angles if possible. A dissection ruler or grid should accompany each photograph.
Using a razor blade and forceps slice the tissue into sections approximately 2 mm wide. Try to slice in a continuous motion, while applying a gentle downward force.
Set aside tissue section and repeat Step 2 as needed. Maintain or keep track of slice order and orientation.
Photograph the sequential 2 mm slices, again using a dissection ruler or grid.
Retain all slices within one container for subsequent processing (lipid removal).