Protocol for vibratome sectioning, immunofluorescence, and S-phase labeling of inner ear organoids

Abstract

Inner ear organoids represent a potentially inexhaustible source of otic tissues, including sensory hair cells and supporting cells, for in vitro manipulation. Here, we present a protocol for labeling S-phase entry of cells in inner ear organoids using 5-ethynyl-2'-deoxyuridine (EdU), followed by fixation and vibratome sectioning. Nuclear EdU is then detected alongside protein markers of interest via immunofluorescence. This workflow enables the visualization of cell and tissue morphologies within developing organoids and assessment of how different manipulations affect cell proliferation. For complete details on the use and execution of this protocol, please refer to Matern et al.¹.

Keywords: Cell Differentiation; Cell culture; Developmental biology; Model Organisms; Molecular/Chemical Probes; Organoids; Stem Cells; Tissue Engineering.

Graphical abstract

Figure 1

Generation of inner ear organoids from mESCs Protocol for stepwise differentiation of mouse embryonic stem cells into inner ear sensory epithelia containing sensory hair cells and supporting cells. ESC, embryonic stem cell. ME, mesendoderm. DE, definitive ectoderm. NNE, non-neural ectoderm. EPI, epidermis. PPE, preplacodal ectoderm. OPV, otic prosensory vesicle. SEV, sensory epithelia vesicle. ‘’ indicates pathway activation and ‘’ indicates pathway inhibition.

Figure 2

Embedding, vibratome sectioning, and immunostaining of inner ear organoids (A) Inner ear organoids in 1× DPBS in a Biopsy Cryomold. (B) Inner ear organoids in a Biopsy Cryomold with 1× DPBS removed. (C) Inner ear organoids embedded in agarose in a Biopsy Cryomold. (D) Agarose block glued to the center of the specimen plate, the side with samples facing upward. (E and E′) One edge of the agarose block parallel to the blade. Blade is positioned at a 15° angle relative to the agarose block. (F) The control panel of the vibratome with preset sectioning parameters. (G) Sectioning of the agarose block. (H–H′′) Isolating individual organoid sections using a biopsy punch. Sections are transferred using cut wide-bore P200 pipette tips precoated in 0.3% PBS-T wash buffer. (I) Isolating and using a mesh cell strainer from a FACS tube cap for immunostaining organoid sections. (J) Illustration of how to aspirate spent buffer during buffer change. (K) Schematic of a microscope slide with two Secure-Seal Spacers. (L) Organoid section punches in the wells of the Secure-Seal Spacers on a microscope slide. (M) Sample sealing with a glass cover slip using forceps.

Figure 3

Immunofluorescence of vibratome-sectioned inner ear organoids (A) Vibratome-sectioned inner ear organoids from DIV3, DIV4, DIV8, and DIV11 stained for SOX2 and the epithelial marker ECAD. Scale bar = 50 μm. (B) Vibratome-sectioned inner ear organoids from DIV16 and DIV21 stained for SOX2 and the hair cell marker MYO7A. Scale bar = 50 μm.

Figure 4

Combined EdU detection and immunofluorescence of vibratome-sectioned organoids Examples of detecting markers of interest using immunofluorescence coupled with Click-iT EdU reaction. In DIV24 organoids treated with 10 μM EdU for 24 h, MYO7A labels sensory hair cells, and SOX2 labels both sensory hair cells and supporting cells. Nuclear EdU marks cells that have gone through the S-phase of the cell cycle within the 24 h period of EdU incubation. Scale bar = 50 μm.

Figure 5

24 h 10 μM EdU and 72 h 0.3 μM EdU does not cause apparent toxicity in inner ear organoid otic structures (A–D) DIV18 and DIV20 organoids (A) treated with 10 μM EdU for 24 h and (B) without EdU treatment. DIV24 organoids (C) treated with 0.3 μM EdU for 72 h and (D) without EdU treatment. Scale bar = 50 μm.