Apr 26, 2026
Fluorescent staining of cells on 3D broccoli-based scaffolds
- Catherine A. Sword1,
- Kristopher Kubow1
- 1James Madison University
Protocol Citation: Catherine A. Sword, Kristopher Kubow 2026. Fluorescent staining of cells on 3D broccoli-based scaffolds. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7ojj8vwz/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 13, 2026
Last Modified: April 26, 2026
Protocol Integer ID: 314931
Keywords: 3D, cell culture, plant-based, decellularized, broccoli, cell biology, bioengineering, fibronectin, scaffold, decellularized broccoli stalk tissue, cells on 3d broccoli, broccoli stalk tissue, fibronectin extracellular matrix of fibroblast, fibroblast, actin cytoskeleton, scaffold, 3d broccoli, based scaffold, cell, extracellular matrix, scaffold production protocol, fibronectin, scaffold production protocol for more information, pore,
Funders Acknowledgements:
Sigma Xi Grant in Aid of Research
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Abstract
This protocol describes how to fluorescently stain the actin cytoskeleton, nuclei, and fibronectin extracellular matrix of fibroblasts cultured on 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 cell culture lab supplies and the reagents listed below, you will require:
- Cover-glass-bottomed dishs or chambers (e.g. Ibidi 80807) for high-resolution imaging of the scaffolds.
- 96-well plates for culturing cells on the scaffolds.
- An inverted confocal microscope for visualizing the scaffolds.
For the proof of concept experiments in the associated manuscript, mouse embryonic fibroblasts (MEF) were cultured in DMEMGibco - Thermo Fisher ScientificCatalog #11995065 + 10 % volume Fetal Bovine SerumGibco - Thermo Fisher ScientificCatalog #16140071 + 1 % volume Antibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 + 1 % volume MEM Non-Essential Amino Acids Solution (100X)Gibco - Thermo Fisher ScientificCatalog #11140050 .
Protocol materials
DAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8417
Acti-Stain 488 PhalloidinCytoskeleton Inc.Catalog #PHDG1
Donkey anti-Rabbit secondary antibody, Alexa Fluor 546Thermo Fisher ScientificCatalog #A10040
MEM Non-Essential Amino Acids Solution (100X)Gibco - Thermo Fisher ScientificCatalog #11140050
DMEMGibco - Thermo Fisher ScientificCatalog #11995065
Fetal Bovine SerumGibco - Thermo Fisher ScientificCatalog #16140071
Antibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
human plasma fibronectinCorningCatalog #354008
16% formaldehydeElectron Microscopy SciencesCatalog #100503-914
Dulbeccos Phosphate-Buffered Saline no clacium no magnesium (DPBS-/-)Gibco - Thermo Fisher ScientificCatalog #14190-144
SaponinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7041-100G
Bovine Serum Albumin (BSA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7030-100G
Gelatin from cold water fish skin, 100gMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7041-100G
Rabbit-anti-human fibronectin polyclonal antibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #F3648
Before start
The procedures involving cell culture were tested with mouse embryonic fibroblasts. Your cell type may require optimization of seeding densities, incubation times, culture conditions, and staining protocol.
Cell seeding and culturing
Seed fibroblasts at the desired density onto 3.5 mm scaffolds as described in the "Seeding and culturing adherent cells on 3D broccoli-based scaffolds" protocol except include 50 μg/ml human plasma fibronectinCorningCatalog #354008 in the final culture medium.
Protocol
CREATED BY
Kristopher E Kubow
Note
The proof-of-concept experiments in the associated manuscript used mouse embyronic fibroblasts (MEFs) seeded at densities between 1,000 and 15,000 cells per 3.5 mm scaffold. To seed 1,000 cells per scaffold, dilute the trypsinized cells to 10,000 cells/ml and prepare a 100 μg/ml fibronectin solution in growth medium. Add 100 μl each of the cell suspension and fibronectin solution to each scaffold. This results in 1,000 cells seeded with 50 μg/ml fibronectin in the medium. Alternatively, seed the cells at the desired density, wait until the cells are attached, and then exchange the medium for medium containing 50 μg/ml fibronectin.
2h 30m
Culture for three days.
Note
Shorter culture times will result in less fibronectin ECM produced.
3d
Fluorescent staining
Prepare 4 % volume formaldehyde (e.g. from 16% formaldehydeElectron Microscopy SciencesCatalog #100503-914 ) in Dulbeccos Phosphate-Buffered Saline no clacium no magnesium (DPBS-/-)Gibco - Thermo Fisher ScientificCatalog #14190-144 .
1m
Prepare the blocking/permeabilization solution: dissolve
- 3 Mass / % volume Bovine Serum Albumin (BSA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A7030-100G
- 1 Mass / % volume
- Gelatin from cold water fish skin, 100gMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7041-100G
- 0.02 Mass / % volume SaponinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7041-100G
in Dulbeccos Phosphate-Buffered Saline no clacium no magnesium (DPBS-/-)Gibco - Thermo Fisher ScientificCatalog #14190-144 .
1m
Aspirate the medium from and add 200 µL sterile PBS to each scaffold.
Note
For this and all aspiration steps, avoid touching the scaffold with the aspirator tip so as to prevent damage to the scaffold. For 6.5 mm scaffolds, which completely occupy the bottom of the well (in a 96-well plate), tilt the well plate and aspirate down to the top surface of the scaffold but do not attempt to push the aspirator tip to the bottom of the well.
5m
Add 200 µL 4% formaldehyde in PBS to each scaffold and incubate at Room temperature for 00:30:00 .
30m
Rinse the scaffolds three times with PBS:
20m
Aspirate the solution from the scaffolds.
Add 200 µL PBS to each scaffold.
Incubate at Room temperature for 00:05:00
Incubate samples with 200 µL blocking/permeabilization solution for 01:00:00 at Room temperature or Overnight at 4 °C .
1h
Prepare the primary staining solution:
- 2 % volume BSA
- 1:300 Rabbit-anti-human fibronectin polyclonal antibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #F3648
in Dulbeccos Phosphate-Buffered Saline no clacium no magnesium (DPBS-/-)Gibco - Thermo Fisher ScientificCatalog #14190-144
1m
Rinse the scaffolds with 200 µL PBS.
5m
Aspirate as much liquid as possible from the scaffolds and add 200 µL primary antibody staining solution to each scaffold.
5m
Incubate samples for 01:00:00 at Room temperature or Overnight at 4 °C .
1h
Prepare the secondary staining solution:
- 2 % volume BSA
- 1:500 Donkey anti-Rabbit secondary antibody, Alexa Fluor 546Thermo Fisher ScientificCatalog #A10040
- 5 μg/ml DAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8417
- 1:400 Acti-Stain 488 PhalloidinCytoskeleton Inc.Catalog #PHDG1
in PBS.
1m
Rinse scaffolds three times with PBS as in Step 7 ( )
20m
Aspirate as much liquid as possible from the scaffolds and add 200 µL secondary staining solution to each scaffold.
5m
Incubate samples for 01:00:00 at Room temperature or Overnight at 4 °C .
1h
Rinse scaffolds three times with PBS as in Step 7 ( )
20m
Use forceps to invert and transfer scaffolds to a cover-glass bottomed dish or chamber (e.g. Ibidi 80807). Add enough PBS 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.
Note
Because of the short incubation times, cells will be be located on the exterior surfaces of the scaffold, primarily on the side facing up during seeding. It is therefore recommended to invert the scaffolds for imaging using an inverted microscope.
5m
Image immediately using an inverted confocal microscope.
Note
For the proof-of-concept experiments in the associated manuscript, we used a Leica Stellaris 8 laser scanning confocal microscope with HC PL APO CS2 20x/0.75 and HC PL APO CS2 40x/1.25 glycerol-immersion objectives. DAPI was excited by a 405 diode laser and emission from 425-505 nm was collected on a HyD S detector. Acti-stain 488 Phalloidin was excited at 498 nm by a white light laser and emission from 505-547 nm was collected on a HyD X detector. The secondary antibody for fibronectin detection (donkey-anti-rabbit Alexa Fluor 546) was excited at 556 nm by a white light laser and emission from 561-814 nm was collected on a HyD S detector. The DAPI and Acti-stain 488 channels were acquired sequentially to avoid bleed-through.