Ds 2mv

Pancreatic sections were processed as described above using antibodies against insulin, glucagon, serotonin, p-S6 and Ki67, and secondary Alexa-Fluor conjugated antibodies (see ESM Methods for further details). For in toto islet immunostaining, 20 islets were handpicked, placed in μ-Slide 8-well plates (80826; Ibidi, Germany) and processed for insulin and serotonin immunostaining, as detailed in ESM Methods. Antibody details are listed in ESM Table 1. Immunofluorescence was examined using an epifluorescence microscope (Nikon 90i; Olympus) and images were taken with a digital camera (Nikon DS-2Mv, Japan). The percentage of beta cells co-expressing insulin and Ki67, p-S6 or serotonin was obtained by dividing the number of positive cells for each staining by the total number of insulin-positive cells in each islet.

Following careful dissection, brains were immediately post-fixed in 10 ml 4% paraformaldehyde (PFA; Alfa Aesar) made up in phosphate buffer (108 mM Na₂HPO₄.2H₂O, 25.3 mM NaH₂PO₄.2H₂O) and shaken for 2 h at 4 °C before being cryopreserved in 20% sucrose (Fisher Chemical, UK) in PBS at 4 °C overnight. The brains were then mounted on a freezing microtome using OCT embedding medium (Thermo Scientific) and 40 µm coronal sections were taken, rostral to caudal. Sections were placed in wells of WHO dimple trays containing 0.01 M PBS. Solution A (0.43 mg ml⁻¹ X-gal, 2% N,N-dimethylformamide, 1 mM MgCl₂; in PBS) was mixed with Solution B (21 mg ml⁻¹ potassium hexacyanoferrate (II) trihydrate, 16.5 mg ml⁻¹ potassium hexacyanoferrate (III); in H₂O) at a ratio of 9:1 to make X-gal staining solution. Following washes in PBS, the sections were incubated in X-gal staining solution at 37 °C in a humidified chamber for 12–48 h. The sections were washed in PBS and then mounted onto slides before being allowed to air dry. The slides were briefly rinsed in water to remove residual salts and then dehydrated in 95% ethanol and 100% ethanol for 2 min each. Slides were placed in Clear-Rite 3 (Thermo Scientific) for 2 min and finally coverslipped using Pertex Mounting Medium (HistoLab). Images were acquired on an Olympus BX41 microscope with a Nikon DS2mv camera attachment.

All the chemicals used were of analytical grade and the aqueous solutions were prepared with deionized (DI) water (resistivity of 18.2 MΩ.cm). Anhydrous n-heptane, CH₂Cl₂, H₂SO₄ (95 %), H₂O₂ (30%), CH₃OH, and C₂H₅OH were procured from Daejung Chemicals & Metals Co., Ltd. of South Korea. Nematic liquid crystal 4-cyano-4’-pentylbiphenyl (5CB) was purchased from Tokyo Chemical Industrial Co., Ltd. of Japan. Malathion, ethion, fenthion, fenobucarb, carbofuran, phosmet, CTAB, phosphate-buffered saline (PBS, pH 7.4), and trichloro(octyl)silane (OTS) were obtained from Sigma-Aldrich, USA. The DNA aptamer with the sequence 5’-ATCC GTCA CACC TGCT CTTA TACA CAAT TGTT TTTC TCTT AACT TCTT GACT GCTG GTGT TGGC TCCC GTAT-3’ was synthesized by Mbiotech (Hanam, Korea).
Polarized optical images of 5CB were captured using a digital camera (DS-2Mv, Nikon, Tokyo, Japan) affixed to a polarizing optical microscope (Eclipse LV100 POL, Nikon, Japan). All images were taken in transmission mode using a 4× objective lens. The gray-scale intensities (GIs) of the optical images were calculated using Adobe Photoshop CC2019 software (Adobe Inc., San Jose, CA, USA). Tests were conducted in at least triplicate for each measurement.

To assess whether there was NOS activity in the egg tissue and where it was located, we used fixation-insensitive NADPH diaphorase staining with nitroblue tetrazolium (Virgili et al., 2001 (link); Müller, 1994 (link)). Eggs were fixed in PBS containing 4% paraformaldehyde for 2 hr at 4°C, followed by cryoprotection in PBS with 12% sucrose for 20 hr. The tissue was soaked in Tissue Tec (Sakura Finetek, Netherlands) for 30 min, frozen, and 10 µm sections were cut on a cryostat microtome (CM3000, Leica, Germany). The sections were incubated for 60 min at 30°C with 50 mmol/l Tris-HCI, pH 7.8, 0.1% Triton X-100, and 0.2 mmol/l nitroblue tetrazolium chloride in the presence or absence (each N = 5) of 0.2 mmol/l β-NADPH to demonstrate fixation-insensitive NADPH diaphorase activity. The sections were dehydrated, mounted with Depex (Serva, Germany) and observed under a compound microscope (Zeiss Axiophot II). Photos were taken with a digital camera (Nikon DS-2 Mv). Since the egg was larger than the field of view of the camera, two pictures had to be taken and were stitched (Photoshop Elements 5, Adobe USA). Contrast and sharpness were optimized.

Cells were observed with a Nikon Eclipse TS100 inverted microscope equipped with the phase-contrast optics, and images were captured using Digital CCD camera system (DS-L1 and DS-2Mv) (all from Nikon instruments Europe BV, Eadhoevedorp, Netherlands). In the experiment measuring cell diameters of NSC, NOP, pOPC, and mixed glial cells, cells were collected after enzymatic dissociation. After making cell suspension, followed by applying the suspension to hemocytometer (Sigma-Aldrich), images were captured using the system mentioned above. Cell diameters of each cell were measured with ImageJ software on basis of the acquired images. Median and confidence intervals (5 and 95% percentile) of each cell type was calculated using Prism 6 software (GraphPad Software Inc., La Jolla, CA, United States) and plotted to determine representative cell diameters and the apparent purity of each cell type.

Image data were acquired with an ArrayScan XTI Live High Content Platform, with a ×20 magnification (Cellomics, Thermo Fisher). For image analysis, 300 validated cells for each treatment group were analysed with Thermo Scientific Co-localisation BioApplication, to obtain the LC3II, LysoTracker and co-localisation fluorescence area per cell (in μm²). Using an epi-fluorescence microscope we acquired representative images (Olympus, BX60, Tokyo, Japan), captured with a digital camera (Nikon DS-2Mv, Tokyo, Japan).
Flow cytometric detection of autophagosomes in cells was performed using a Cyto-ID Autophagy Detection Kit (Enzo Life Science, New York, NY, USA). After treatments, cells were collected by centrifugation and resuspended in 1 × assay buffer. CYTO-ID Green stain solution was added to each sample, then incubated for 30 min at 37°C in dark. After washing the cells with 1 × assay buffer, data were acquired using a CytoFlex Flow Cytometer (Beckman Coulter) and analysed with CytExpert 2.0 Software (Beckman Coulter). Cells were first gated for viable cells (FSC-A vs SSC-A). Cells were then gated to exclude apoptotic cells (FSC-A vs FSC-H). Using Cyto-ID fluorescence in the FITC-A channel, autophagic vesicles were quantified and plotted as cell counts in superimposed histograms.