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Tissue expansion microscopy (TissUExM) allows super-resolution imaging by physically expanding biological samples. Here, we present a protocol for ultrastructure expansion microscopy of Xenopus embryos using TissUExM. We describe steps for tissue fixation, embedding, fluorescence labeling, and expansion. We demonstrate that our protocol provides enhanced resolution for studying subcellular structures, such as centrioles and cilia. This protocol has the potential to offer a broad range of applications to decipher sub-cellular organization in Xenopus embryos.
Figure 1. Gelation chamber preparation, gelation set up and gelation
(A) Schematic representation of the gelation chamber.
(B) Gelation chamber with embryos positioned just before gelation.
(C) The gelation set up is installed under a fume hood and includes: PBS (1); APS and SDS (optional 4OH-TEMPO) on ice (2); cooling block for iMS solution (3); gelation chamber (4); coverslips placed on a support to be easily accessible (5) and pipettes preset to the volumes required for preparation of the activated gelation solution (6).
(D) Schematic representation of embryo positioning in the gelation chamber and gelation procedure.
Figure 2. Measurements to evaluate expansion factor and gel trimming
(A) Embryo after fixation.
(B) Gel on day 8 after equilibration. At this stage the 12 mm initial gel was expanded by a factor 2.
(C) Gel on day 8 after trimming at 1 mm around the embryo. In this example the length of the trimmed rectangle is 12 mm.
(D) Gel on day 14 before imaging. The trimmed gel measured 24 mm and was thus expanded by a factor 2. The total expansion factor is calculated by adding the factors for the two expansion stages, giving a final value of 4X.
Figure 3. Schematic representation of embryo mounting before observation
Figure 4. Multiciliated cell nanoscale organization revealed by TissUExM on Xenopus embryos
(A) Confocal field of view of Xenopus embryo epidermis stained for Acetylated-α-Tubulin.
(B) Expanded Xenopus embryo epidermis stained for Acetylated-α-Tubulin imaged with the same confocal parameters as in A.
(C) Zoom on expended cilia stained with Acetylated-α-Tubulin.
(D) Quantification show minimal deviation in cilia diameters (Mean= 293.12 nm, SD= 29.51 (10%), n=68 cilia from two independent experiments).
(E) Confocal field of view of Xenopus embryo epidermis stained for distal appendages (DA, Cep164, green) and centrioles (Centrin, red).
(F) Expanded Xenopus embryo epidermis stained for distal appendages (DA, Cep164, green) and centrioles (Centrin, red) imaged with the same confocal parameters as in E.
(G) Top (top) and lateral (bottom) views of basal bodies from expanded Xenopus embryo epidermis stained with Acetylated-α-Tubulin.
(H) Same pictures as in G indicating where the diameter x (Dx), diameter y (Dy), diameter (D), and length (L) were measured.
(I) Dx/Dy and L/D ratio quantification. Average is 1.06 for Dx/Dy and 1.89 for L/D. Data are represented as mean ± SD. (n=30 centrioles from two independent experiments).
Scale bar A, B: 10 μm; C: 2.5 μm, E, F , G, G’: 1 μm.
