Vs120 virtual slide system

Images were acquired using a VS120 Virtual Slide System (Olympus). Whole slides were first scanned in the DAPI channel (Excitation (Ex): 388 nm; Emission (Em): 448 nm) at 2× magnification and then the outlines of whole coronal tissue sections were traced for scanning at 40× magnification. A focus-map (the highest density possible) was auto-generated across the entirety of each coronal section and the plane of focus was automatically determined based on the DAPI channel. DAPI (Ex: 388 nm; Em: 448 nm), DsRed (Ex: 576 nm; Em: 625 nm), and GFP (Ex: 494 nm; Em: 530 nm) signals were imaged in the same focal plane. Optimum exposure times were initially determined manually and then kept consistent for all images (DAPI, 50 ms; DsRed, 100 ms; GFP, 50 ms). The .vsi files generated were approximately 2 GB each in size.

Developing rice seeds, Yamadanishiki, Nipponbare, and Gohyakumangoku (sampled in 2014 NRIB) at 17 DAF were examined with immunofluorescence microscopy. For immunofluorescence staining, seed sections (approximately 4 μm thick) embedded in paraffin were deparaffinized with xylene followed by a stepwise change of ethanol. The slides were exposed to 1% hydrogen peroxide/methanol for 30 min to block endogenous peroxidase activity before being washed with water and Tris-buffered saline (TBS). The sections were incubated with anti-glutelin antibodies (1/100 diluted) or an anti-TOC75 antibody (1/100 diluted) overnight. As secondary antibodies, we used Alexa Fluor 594 donkey anti-rat IgG (against GluB-4/5), Alexa Fluor 488 donkey anti-rabbit IgG (against GluA, GluB-1, GluC, GluD, and TOC75), or Alexa Fluor 488 donkey anti-rat IgG (against GluB-4/5) at a 1:50 dilution. The sections were treated with SlowFade Gold Antifade reagent with DAPI (Molecular Probes). Immunofluorescence microscopy analysis was performed using a BX-52 microscope (OLYMPUS, Tokyo, Japan). Images of whole sections of rice grain were captured using a VS120 virtual slide system (OLYMPUS) and analyzed using an OlyVIA software (OLYMPUS). Tissue assignment of the immature rice grain was referred to a previous report (Wu et al. 2016 (link)).

Harvested organs were immediately placed in 10 mL of 10% (v/v) neutral buffered formalin (VWR, Radnor, PA, USA) and kept on ice until transferred to a 4 °C cold room, where organs were fixed overnight on a rocker. Organs were rinsed with 1× PBS, pH 7.4, for 10 min (3×) before embedding in paraffin or Tissue-Plus OCT (Thermo Fisher Scientific, Waltham, MA, USA). For histology, organs were cut in 6 µm serial sections using a CryoStar NX70 (Thermo Fisher Scientific, Waltham, MA, USA) and stained with MitoSox or H&E (Thermo Fisher Scientific, Waltham, MA, USA), or Trichrome (Polysciences, Warrington, PA, USA). Histological differences were quantified using ImageJ software. H&E and Trichrome images were collected by an Olympus VS120 virtual slide system (Olympus Life Sciences, Waltham, MA, USA). MitoSox images were collected with a Eclipse Ts2 fluorescent microscope (Nikon, Melville, NY, USA). Mean technical replicates (3–6 per parameter) were used to represent effects on a single heart. A minimum of three hearts were used for all quantified parameters. Protein quantification experiments used corresponding organs from seven different wildtype male, wildtype female, p66Shc^−/+ male, or p66She^−/+ female fish for a total of N = 7 per quantification.

P0 mouse brains were dissected and immediately fixed in 4% paraformaldehyde at 4°C overnight. Sucrose-dehydrated tissues were embedded in Tissue-Tek O.C.T cryotissue-embedding compound (Sakura). Sagittal sections (15-μm thick) prepared by cryostat sectioning were labeled with fluorescent RNAScope probes recognizing Ube4b, Vgat and Vglut2. The whole procedure followed the User Manual of the RNAScope Multiplex Fluorescent Reagent Kit v2 Assay (ACD, 323120). Images were acquired with the Olympus VS120 virtual slide system (10× and 20× tile scan) and processed with MetaMorph software.

For the histological analyses, heart tissue at the age of 12 weeks was fixed in paraformaldehyde, 4% in phosphate-buffered saline, embedded in paraffin, sectioned, and subjected to hematoxylin–eosin staining and Masson’s trichrome staining. Stained sections were photographed using Virtual Slide System VS120 (Olympus Scientific Solutions, Tokyo, Japan).