Hematoxylin solution

Embryos were sampled once a day from 1 to 15 dpf and fixed in Bouin’s solution for 48 h before being washed and conserved in 70 % alcohol. In order to facilitate their cutting orientation, embryos were first placed in 1.5 % agarose blocks. These blocks were then dehydrated in ascending series of ethanol (70–100 %) before being embedded in paraffin. Four micrometer sections were performed with a Leitz Wetzlar microtome and collected on glass slides. Hematoxylin and Eosin staining were performed according to a protocol adapted from Gabe [36 ] as follows: Eosin Y (Sigma-Aldrich, Saint Quentin Fallavier, France) was diluted in water at 1 % and used from 30 s to 1 min; Hematoxylin solution modified according to Gill III (Merck, Darmstadt, Germany) was used from 5 to 10 min. Stained sections were examined using a light upright optical microscope (Nikon Eclipse Ni-U) at 10× and 40× magnifications (Nikon France, Champigny-sur-Marne, France). In total, 45 embryos from 11 spawn have been used for the histological study. Three embryos were transversally cut per day from 4 to 15 dpf. In addition, three embryos were longitudinally cut at 5, 10 and 15 dpf.

Spheroids were washed once with PBS and transferred into an agarose bedding (4% (w/v) agarose in ddH₂O). Spheroids were then stained with hematoxylin solution (Merck, Darmstadt, Germany) to identify spheroid locations. Spheroids were then incubated with 4% PFA solution for 10 min at room temperature and, after removal of supernatant, covered with 1% agarose. Agarose cores were transferred into 4% PFA in embedding cassettes. Samples were dehydrated and embedded in paraffin. In total, 3 μm slices were cut with a rotary microtome (RM 2255 Mikrotom, Leica Biosystems, Wetzlar, Germany) and sections were transferred to glass slides before they were incubated at 56 °C overnight, prior to storage. Sections were deparaffinized with Neo-Clear solution (Merck, Darmstadt, Germany) for 30 min at room temperature and rehydrated with decreasing concentrations of ethanol (100%, 96%, 70%) and H₂O (3 min each). Next, samples were incubated with Dako target retrieval solution pH 6 (Dako, Agilent Technologies, Santa Clara, CA, USA) for 30 min inside a steam cooker and afterwards washed two times for 3 min with 1 × TBST (Tris-buffered saline with 0.1% (v/v) Tween-20, Carl Roth Karlsruhe, Germany). Treatment with peroxidase blocking reagent (Dako, Agilent Technologies, Santa Clara, CA, USA) and staining steps were conducted as described above.

Tissue fixation was conducted by immersing samples into a formaldehyde solution (Merck, Darmstadt, Germany). Tissue samples that became histopathic were immersed in xylol. The samples were then immersed in a series of alcohol, namely 100%, 90% and 70% ethanol (Merck, Darmstadt, Germany). Next, the samples were soaked in distilled water and soaked in a hematoxylin solution for 5 min (Merck, Darmstadt, Germany). The sample was then placed under running water until the blue color on the slide disappears and the only blue color left was on the colored organ. The next stage included staining the tissue sample with eosin to color the cell cytosol in it (Merck, Darmstadt, Germany).
The sample then entered a multilevel alcohol immersion stage with concentrations of 70%, 95% and absolute alcohol (100%), respectively. Next, the slides were wiped using tissue on parts that were still wet to make sure they were completely dry. Following that, it was dipped into 3 xylol tubes. Finally, the tissue samples were covered with slide cover.

Bafilomycin A1 (Baf), 3-methyladenine (3-MA), chloroquine (CQ), and 5-Bromo-2-deoxyuridine (BrdU) were purchased from Sigma Aldrich (St Louis, MO, USA). Antibodies against p62 and LC3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against autophagy-related protein 5 (ATG5), ATG7, dystrophin, beclin1, and lysosome-associated membrane protein 2 (LAMP2) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Hypoxia-inducible factor (HIF)-1α, CD31, and BrdU antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Hematoxylin solution and the CYTO-ID kit were purchased from Merck (Darmstadt, Germany) and Enzo Biochem (Farmingdale, NY, USA), respectively.

Paraffin embedded sections were first heated in an oven at 65°C for 1 h before undergoing several series of washes of xylene and ethanol to deparaffinize/rehydrate. After deparaffinization/rehydration, the slides were immersed in Hematoxylin solution (Merck) for 3 min. The slides were rinsed with water, and counterstained with 0.5% Eosin G-solution (Merck) for 5 min. After 30 s of rinsing in water, the slides were dehydrated in series of increasing ethanol and xylene concentrations. The coverslips were mounted on the slides with Entellan Neu mounting medium (Merck).