Apr 10, 2026
Staining 3D broccoli-based scaffolds for microstructure visulaization
- Catherine A. Sword1,
- Kristopher Kubow1
- 1James Madison University
Protocol Citation: Catherine A. Sword, Kristopher Kubow 2026. Staining 3D broccoli-based scaffolds for microstructure visulaization. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyobeegx9/v1
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: April 10, 2026
Last Modified: April 10, 2026
Protocol Integer ID: 314771
Keywords: 3D, cell culture, plant-based, decellularized, broccoli, cell biology, bioengineering, fibronectin, scaffold, decellularized broccoli stalk tissue, broccoli stalk tissue, staining 3d broccoli, microstructure of scaffold, scaffolds for microstructure visulaization, scaffold, based scaffold, 3d broccoli, calcofluor white stain, scaffold production protocol, microstructure visulaization, scaffold production protocol for more information, pore, isotropic arrangement of pore, microstructure, calcofluor,
Funders Acknowledgements:
Sigma Xi Grant in Aid of Research
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Abstract
This protocol describes how to use the calcofluor white stain to visualize the microstructure of scaffolds derived from decellularized broccoli stalk tissue. It is part of a protocol collection describing how to produce and use the plant-based scaffolds for short-term (days), in vitro, cell-culture experiments. The scaffolds are composed of an isotropic arrangement of pores (median diameter, 70 μm), and have a stiffness in the range of mid-stiffness human soft tissues. Please see the scaffold production protocol for more information.
Materials
In addition to standard lab supplies and the reagents listed below, you will require:
- Cover-glass-bottomed dish or chamber (e.g. Ibidi 80807). A plastic well-plate will also work but only for low-magnification objectives (usually not higher than 10x) and may introduce background fluorescence.
- An inverted fluorescence microscope with the ability to visualize DAPI stain. For example, on a widefield microscope use 392/22 nm excitation and 447/60 emission filters, and on a laser scanning confocal microscope excite with a 405 nm laser and collect emission in the range 425-441 nm. These ranges are just examples and other similar settings will also work.
Protocol materials
Calcofluor WhiteHoneywell FlukaCatalog #18909
potassium hydroxideAcros OrganicsCatalog #42414
Before start
This protocol is optimized for 3.5 and 6.5 mm scaffolds. This procedure is not compatible with cells (alive or fixed). It is not necessary to perform this protocol under sterile conditions.
Transfer desired number of scaffolds to a cover-glass-bottomed dish or chamber (e.g. Ibidi 80807). Plastic dishes such as well plates may be used but produce higher background fluorescence and are generally not compatible with microscope objectives > 10x.
1m
Pipette 100 µl each of Calcofluor WhiteHoneywell FlukaCatalog #18909 and 10% w/v10 Mass / % volume potassium hydroxideAcros OrganicsCatalog #42414 onto each scaffold.
1m
Incubate at Room temperature for at least 00:10:00 to allow the stain to penetrate and bind to the scaffold.
10m
Rinse the scaffolds once with deionized water.
1m
Add enough deionized water to the scaffolds to keep them hydrated, but not so much that the scaffolds float above the dish/well surface. Having the scaffolds stationary on the surface will make them easier to image.
1m
Transfer the dish/chamber/plate to an inverted fluorescence microscope and examine using the same settings used to examine DAPI. For example, on a widefield microscope use 392/22 nm excitation and 447/60 emission filters, and on a laser scanning confocal microscope excite with a 405 nm laser and collect emission in the range 425-441 nm. These ranges are just examples and other similar settings will also work.