High-throughput Imaging and Analysis Workflow for Evaluating Skin Cell Phenotypes and Proliferation States in Tissue Samples

Summary

The combination of iterative-bleaching-extends-multiplexity (IBEX) and a commercial nucleotide labeling assay (Click-iT EdU) enables the detection and categorization of dividing cell types in highly dynamic processes in fixed frozen murine tissue sections. Furthermore, a novel open-source image processing pipeline provides high-throughput image acquisition and analysis.

Abstract

Skin injuries initiate a complex regenerative cascade that engages various cell types. Amongst those, peripheral glial cells play a critical role in ensuring the success of the repair process. However, our understanding of these processes remains incomplete due to limitations in current technologies. Therefore, the iterative-bleaching-extends-multiplexity (IBEX) method was employed to characterize changes to the cellular composition of the skin using antibodies directed against proteins expressed by key cell types and structures involved in tissue regeneration. Importantly, antibodies directed against cell proliferation markers often underestimate the number of dividing cells in the skin. To overcome this limitation, Click-iT EdU chemistry was combined with the IBEX method, allowing detailed characterization of proliferating cell types. Using a fully automated spinning disk confocal microscope, a high-throughput workflow was developed for iterative imaging of samples sectioned into 24-well plates. Beyond extending the IBEX method to evaluate cell states in situ with Click-iT EdU, a novel open-source image processing pipeline was also introduced to perform image correction, stitching, and registration, and can be used by researchers without extensive programming experience. Moreover, a detailed documentation on processing and visualizing highly multiplexed imaging data (using conventional image analysis software, including Fiji and QuPath) is also provided. In summary, this protocol highlights the versatility of the IBEX protocol and demonstrates its adaptation to an established conventional imaging platform. Notably, combining IBEX with Click-iT EdU labeling enables the detection and categorization of actively dividing cell types in intact tissues and during highly dynamic processes at spatial resolution.

Introduction

The skin is the largest organ of the human body and the primordial barrier to protect the body from the outside environment¹. Consequently, it is exposed to a plethora of external challenges that compromise its barrier function. Therefore, the skin has developed complex mechanisms to restore tissue integrity and functionality following damage. Skin wound healing takes place in three overlapping stages: inflammation, proliferation, and maturation/remodeling. These three stages involve several different cell types, such as dermal fibroblasts and immune cells, that act together to restore the initial biological properties of the injured skin tissu....

Protocol

​All animal breeding, housing, and experimentation were conducted according to the guidelines of the veterinary office of the Canton of Zurich, Switzerland.

1. Full-thickness skin wounds (Day 0)

1. Induce 8-10 weeks old mice with 5% isoflurane in 70% O₂ and maintain it using 2.5% isoflurane.
2. Shave the back skin and clean it thoroughly.
3. Disinfect prior to the surgery using antibacterial soap and 70% EtOH solution.
4. Apply preoperative analgesia using a subcutaneous injection of buprenorphine (30 µg∙mg∙kg⁻¹).
5. Generate four circular full-thickness....

Results

This method allows adequate detection of proliferating cell types by combining EdU Click-iT chemistry with IBEX in fixed frozen murine skin sections. Detection of proliferating cell types was compared using different methodologies, in particular, labeling of proliferating cell types with Ki-67 antibody and detection of cell proliferation using the EdU Click-iT chemistry approach (Figure 1). We observed that Ki-67 antibody labeling underestimates the number of.......

Discussion

Cell proliferation is one of the four main stages of skin repair, and the transition between the inflammatory phase and the proliferative phase is critical for a successful repair process²⁰. A well-regulated immune response is essential, as prolonged inflammation can lead to chronic wounds, a significant global health issue^1,21. Proliferation is a key biological process, not only important for tissue repair but also for organism developmen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies and reagents	SOURCE	IDENTIFIER	Comments
Antibodies
CD31 Alexa Fluor 647 (Cycle 2)	BioLegend	Cat#102516	1:100
AB_2161029
CD45 Alexa Fluor 488 (Cycle 2)	BioLegend	Cat#103122	1:100
AB_493531
Ki-67 FITC (Cycle 1)	BioLegend	Cat#151212	1:50
AB_2814055
Ki-67 chicken (ck)	LSBio	Cat#LS-C772946	1:800
Ki-67 rat (rt)	BioLegend	Cat#652402	1:100
AB_11204254
Neurofilament H&M (NF-H/NF-M) Alexa Fluor 647 (Cycle 3)	BioLegend	Cat#837710	1:300
AB_2734613
p75NTR (Cycle 1)	Cell Signaling Technology	Cat#8238                       AB_10839265	1:1500
Vimentin Alexa Fluor 488 (Cycle 3)	BioLegend	Cat#699305	1:100
AB_2888889
Alexa Fluor 647 donkey anti rabbit	Jackson ImmunoResearch	Cat#711-605-152
Reagents
Cellvis 24 well Glass Bottom Plate	Cellvis	P24-1.5H-N
Chrome alum gelatin	Newcomer Supply	Cat#1033A
Click-iT EdU kit	Thermo Fisher Scientific	C10637
Hoechst 33342	Sigma-Aldrich	Cat#14533; CAS# 23491-52-3
LiBH4, Lithium Borohydride, 95%	Thermo Fisher Scientific	Cat#10438443
Tissue-Tek O.C.T Compound Sakura	Biosystems Switzerland	Cat#7109-4583
PBS	Thermo Fisher Scientific	Cat#10010-015
Superfrost Plus Adhesion Microscope Slides	Epredia	Cat#J1800AMNZ
Triton X-100	Sigma-Aldrich	Cat#T8787
Equipment
Hair brush	Faust AG	Cat#9172051 and 9172050
Microtome blades	Biosystems Switzerland	Cat#207500011
Microscope	Molecular Devices, ImageXpress Confocal HT.ai
Oven	Thermo Fisher Scientific	Cat#HBMCR4220
Rocker shaker	Edmund Bühler GmbH	Cat#32310015