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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster H16 - Optimizing Multiplexing of Antibodies for Immunocytochemistry

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Dennis Ngo (he/him/his)

Undergraduate Student
Johns Hopkins University
Springfield, Virginia, United States

Co-Author(s)

HO

Huan Ting Ong, PhD (she/her/hers)

Research Fellow
Mechanobiology Institute at the National University of Singapore, Singapore

Primary Investigator(s)

JY

Jennifer L. Young

Principal Investigator
Mechanobiology Institute at the National University of Singapore, United States

Introduction: Immunocytochemistry (ICC) multiplexing involves sequential 4-channel imaging of different immunostains, with several rounds of 4-color immunofluorescence, followed by stripping of fluorophores, and restaining for new antigens. Traditionally, ICC multiplexing has been employed to examine tissue samples and various monolayered culture samples, however its application to study extracellular matrix (ECM) proteins in cardiac fibroblasts remains unexplored. Two rounds of ICC multiplexing on young and aged cardiac fibroblasts cultured on different ECM coatings were performed to optimize ICC multiplexing of ECM proteins produced by cardiac fibroblasts and to compare the production of seven different ECM proteins within the same cell culture samples produced by young vs. aged cardiac fibroblasts. Distinct stain groups were utilized during the multiplexing staining process, featuring different combinations of staining the seven ECM proteins in the two rounds of multiplexing. Photobleaching with 4.5% H2O2/H2O and an LED light source was effective for every stain used for GFP and RFP channels, but was ineffective for 1° rabbit anti-fibronectin, 2° goat anti-rabbit 633. It is concluded that (i) ICC multiplexing is an effective technique for producing comprehensive images of ECM composition and architecture, and (ii) photobleaching with 4.5% H2O2/H2O and an LED light source proves effective for most immunofluorescent stains, but specific adjustments are needed for certain antibodies like rabbit anti-fibronectin and goat anti-rabbit Alexa Fluor 633.

Materials and

Methods: Primary cardiac fibroblasts were harvested from young and aged mice. Cells were cultured on 18 mm coverslips placed in 24-well plates. Coverslip coatings included collagen, fibronectin, and poly-D-lysine (PDL). Solutions for cell culture and immunocytochemistry (ICC) were also prepared, including cell expansion and growth media, accustase, 4% paraformaldehyde (PFA)/PBS, and 4.5% H2O2/H2O and an LED light source for photobleaching (20,000 lux/12V). Dyes and stains used include Hoechst, phalloidin, α-SMA, collagen types I and III, paxillin, fibronectin, and integrin α5 along with a blocking buffer of 1% bovine serum (BSA)/PBS.

Coverslips were coated with collagen, fibronectin, or PDL. Young and aged cardiac fibroblasts (YCF and ACF) were thawed, mixed with expansion media, centrifuged, and the supernatant was replaced with normal cell media. After an incubation period, cells were plated in flasks with cell media and incubated for 3 days. Post incubation, media was changed, and cells were detached for seeding on the coated coverslips. YCF were seeded at 30,000 cells/well and ACF at 20,000 cells/well in 1 mL of media, with media changes every 2 days.

Once confluency was reached, cells were fixed and permeabilized. Cells were blocked with 1% BSA/PBS before primary antibodies were applied and incubated. Cells were then incubated in secondary antibodies, including Hoechst. Imaging was conducted using an EVOS™ M5000 Imaging System. Photobleaching was performed with 4.5% H2O2/H2O for 1 hour 45 minutes under LED light, followed by a second round of primary and secondary antibody application, staining, and imaging.

Results, Conclusions, and Discussions: The fibronectin stain was unable to be bleached after 1 hr 45 minutes in 4.5% H2O2/H2O under LED light at 20,000 lux/12V, most likely due to the high photostability of the goat anti-rabbit Alexa Fluor 633. A future study should attempt to photobleach this stain with a longer time in hydrogen peroxide under an LED, ensuring that there is excess hydrogen peroxide solution submerging the sample. This is an interesting result, as previous literature has reported the photo-inactivation resistance of Alex Fluor 546, Alex Fluor 568, and Alex Fluor 594, but Alex Fluor 633 has not been mentioned. Further testing is required to confirm whether the primary or secondary antibody used to stain fibronectin resisted photo-inactivation. We recommend staining fibronectin in the final round of multiplexing to avoid carryover signal in subsequent rounds of staining. It is also interesting to note that conjugated F-actin 546 bleached effectively, demonstrating that conjugated antibodies work well with this project’s multiplexing protocol. We can also see the latter stain with fibronectin in round 2 produced a clear image of the cell morphology.

The application of ICC multiplexing to study extracellular matrix (ECM) proteins in cardiac fibroblasts has the potential to elucidate significant differences in the ECM composition between young and aged fibroblasts. This distinction is crucial for understanding the mechanisms underlying cardiac aging and the progression of cardiac fibrosis. Our study highlights a protocol that may unveil major differences in aged cardiac fibroblasts compared to young by allowing for the creation of comprehensive images of ECM proteins within the same cell culture samples. By employing multiple rounds of staining and imaging, we have been able to analyze a plethora of specific proteins within the same cell culture samples, potentially offering insights that can be obscured in traditional one round 4-channel staining experiments. We have also highlighted the ECM proteins and antibodies that were either effective or ineffective with our photobleaching protocol, which may aid in future experimental design.

Acknowledgements (Optional): I wish to thank Dr. Huan Ting Ong for acting as my incredible primary mentor; Faris Muhammad for introducing me to the Mechanobiology Institute’s facilities; Raageshwari Arulselvam and Anmadusha Hengst for providing necessary materials for the project; Martin Kiwanuka for guidance on MDA-MB-231 human cancer cells for preliminary photobleaching testing; Hepi Susapto and Avery Sun for providing their expertise on antibody chemistry; Soleil Su and Han Vu for being helpful peers throughout my time at the Mechanobiology Institute; and Dr. Jennifer Young for giving me the opportunity to work at the Mechanobiology Institute and providing invaluable research advice.