80i light microscope

For the purpose of histological analysis, mice were sacrificed by cervical dislocation and eyeballs were dissected at 14d, 21d and 28d after modeling. After a fixation in 4% paraformaldehyde for 24 h, the tissues were dehydrated, dipped in wax, embedded, and sliced in sequence. The slices were under a routine operation of dewaxing and then HE staining was used for pathological examination. Photos were taken by Nikon 80i light microscope (Nikon, Sendai, Japan) and analyzed in terms of corneal thickness.

For the differentiation of NSCs, cells were plated on poly-d-lysine-coated cover slips in DMEM/F12 medium with 1% FBS ( Invitrogen, Carlsbad, CA, USA) instead of growth factors for 7 days. Cells were washed in PBS three times and fixed in 4% formaldehyde for 10 min. Triton X-100 (0.2%) was used for permeabilization for 10 min at room temperature. Then, the cells were blocked in 1% BSA (Solarbio, Beijing, China) in PBS for 20 min at room temperature. Next, the cells were incubated with primary antibodies (rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) 1:1000, Abcam, Cambridge, MA, USA, and mouse monoclonal anti-neuronal class III β-Tubulin (Tuj1) 1:500, Abcam, Cambridge, MA, USA) overnight at 4 °C. After washing with PBS, the cells were incubated with secondary antibodies (Alexa Fluor 488-conjugated IgG 1:1000 and Alexa Fluor 594-conjugated IgG 1:1000, Invitrogen, Carlsbad, CA, USA) in PBS for 40 min at 37 °C. Images were captured using a Nikon 80i light microscope equipped with a CCD camera.

For histological staining, intestinal organoids were fixed with the same protocol as used for immunohistochemistry, sliced in 4 µm thick sections and rehydrated in PB three times for 5 min each. For hematoxylin-eosin (HE) staining the slides were stained in Mayer’s hemalum for 7 min, followed by rinsing in running tap water for further 7 min and staining with 0.1% (v/v) eosin [addition of 0.01% (v/v) glacial acetic acid] for 5 min. For Periodic Acid Schiff (PAS) staining, the rehydrated slides were transferred into 1% (w/v) periodic acid solution for 10 min, followed by rinsing in tap water for 10 min and in distilled water for 2 min twice. Further staining with Schiff’s reagent for 15 min was followed by washing in 35°C warm tap water before the slides were stained with Mayer’s hemalum for 5 min and rinsed in running tap water for 10 min. After HE and PAS staining, the slides were transferred into staining dishes with increasing ethanol concentrations [70% < 80% < 96%, (v/v)] for a few seconds each. Finally, the slides were treated with Roti-Histol for 3 min twice and mounted with Roti-Histokit. All staining reagents were obtained from Carl Roth GmbH, Karlsruhe, Germany. Pictures were taken with the Nikon 80i light microscope.

Liver and kidney tissues were fixed in 10% neutral formalin solution for 24 h, dehydrated in ascending series of ethanol, cleared in xylene then embedded in paraffin wax. Sections were stained with conventional haematoxylin and eosin (H&E) dye and examined using a Nikon 80i light microscope (Nikon Corporation, Tokyo, Japan).

Arthrospira, O9.13F and four strains PCC 8005, PCC 7345, SAG 49.88 and CCALA 023 were cultivated without shaking under cold room conditions in the Zarrouk medium under a constant lighting of 10 µmol photons m⁻² s⁻¹ with a daily amplitude of 5–8 °C for 10 months. After 3 and 10 months of cultivation in the cold room, all samples were inoculated onto the solid Zarrouk medium and grown at 20 °C under a light intensity of 20 µmol photons m⁻² s⁻¹ for two months. The photographic documentation was made using a Nikon 80i light microscope.