Eosin y

Paraffin-embedded tissue sections (5 µm) were prepared on a microtome (Leica), dried overnight at room temperature and de-paraffinized using xylene and a descending alcohol dilution. The sections were incubated in 1% (v/v) acetic acid for 20 s before staining with hematoxylin (modified after Gill, Merck) for 10 min. Counter-staining with eosin Y (Merck) was carried out after two consecutive washes with warm tap water for 2 min. Then, the sections were dehydrated using an ascending alcohol series and two incubations with xylene before embedding in Eukitt embedding medium (Merck).

For histochemistry and immune‐histochemistry, paraffin‐embedded skin samples were cut into 4 μm sections. Haematoxylin and eosin (HE) staining using Mayer's Hemalaun (1.09249.2500, Merck) and Eosin Y (1.15935.0100, Merck) was done in a linear slide stainer (Leica ST4040). For Masson–Goldner trichrome staining, kits were used according to the manufacturer's recommendations (12043, 14604, Morhisto). Automatic scanning of full slides in 40x magnification was done using the VS‐120‐L Olympus slide scanner 100‐W system and processed using the Olympus VS‐ASW‐L100 program. Evaluation of the epidermal thickness and murine vessel quantification was done according to published work (Ebner‐Peking et al., 2021 (link)), both measured in the Olympus VS‐ASM‐L100 program. Per group, four biological and at least three technical replicates were included.

For hematoxylin and eosin (HE) staining, one serial section from each set was dewaxed in xylene (2 × 15 min), rehydrated through ethanol series with abs. 100%, 99%, 90% and 70% (5 min each) and rehydrated completely in sterile water. Hydrated sections were stained in Mayer’s hematoxylin (Wako, Japan) for 10 min, rinsed in water for 5 min, then stained with eosin Y (Merck, Germany) for 5 min, and further rinsed in water for 30 sec. The stained sections were dehydrated through the same ethanol series in reverse with agitation (few sec each), cleared by xylene (2 × 5 min) and finally mounted in Entellan mounting medium (Merck, Germany). HE stained sections were observed and recorded using an ECLIPSE Ni microscope (Nikon, Japan) and BIOREVO BZ-9000 microscope (KEYENCE, Japan). All sampled corals appeared visually healthy at the time of collection and tissues displayed normal cell morphology, including no signs of fragmentation, wound repair or necrosis (as per criteria in^{41 (link)}). The number of CAMA were counted into two categories, basophilic and eosinophilic CAMAs. The area of whole tissue (including skeleton region) was measured by tracing the outer edge of the tissue section using Fiji software^{64 (link)}.

Samples were stained with Mayer’s hematoxylin (Merck, Darmstadt, Germany). for 10 min and washed with tap water for 10 min. Next, samples were stained with 1% alcohol Eosin Y (Merck) for 10 s, and then dehydrated with 70 and 95% ethanol once each and 100% ethanol twice. Slides were then mounted using a permanent mounting medium (Permount Mounting Medium, Electron Microscopy Sciences, Hatfield, PA, USA).

The anesthetized newts were perfusion-fixed with 4% paraformaldehyde in PBS, and subsequently the head portion was immersion-fixed with 75% methanol/25% acetic acid fixative at 4°C overnight. Heads were decalcified in Osteosoft (Merck, Darmstadt, Germany) and embedded in paraffin. Serial paraffin sections of 10 µm thickness were made from the brains of intact newts and newts at different times after the surgery, and stained with HE. Hematoxylin (Muto Pure Chemicals, Tokyo, Japan) diluted 1:10 and Eosin Y (Merck) were used for the staining.

Tissues were fixed with 10% neutral-buffered formalin (Dana Korea, Incheon, Korea), dehydrated with increasing concentrations of ethanol (70% to 100%), cleared with xylene (Duksan, Ansan, Korea), and followed by paraffin infiltration (Merck, Darastadt, Germany). Thereafter, tissues were embedded in paraffin and sectioned (6 μm) using a rotary microtome (Leica, Wetzlar, Germany). Sectioned slides were deparaffinized and stained with hematoxylin (YD Diagnostics, Yongin, Korea) and eosin Y (Merck, Darastadt, Germany). Images were examined by light microscopy (Leica, Wetzlar, Germany) and rendered by Leica software.