Infinity 3

Calcofluor white staining, which stained both non-crystalline and crystalline cellulose, is a method commonly used for estimating cellulose abundance, including dinoflagellate cells which had no callose (Kwok and Wong, 2003 (link); Fujise et al., 2014 (link)). Ecdysal cysts were first fixed in 2% (w/v) paraformaldehyde overnight at 4°C before replacing with PBS containing 10 μg ml⁻¹ CFW. Photomicrographs were taken from a Leica fluorescent microscope (DMLS) equipped with a digital camera (INFINITY 3, Lumenera).
In order to visualize the localization of LpCesA1p within the internal cell wall, cryosections (5 μm) were prepared as described previously (Soyer-Gobillard et al., 2002 (link)), except that infiltration of cell pellets with polyvinylpyrrolidone (PVP)/sucrose (20% (w/v) PVP, 1.7 M sucrose) was performed by a stepwise manner (Ausseil et al., 1999 (link)). Confocal imaging employed a Leica TCS SP5 II confocal system. Whole cell immuno-labeling was performed as described previously (Soyer-Gobillard et al., 2002 (link); Kwok and Wong, 2010 (link)).

Semi-thin sections were observed with an Olympus BX60 microscope (Tokyo, Japan) between crossed-polarizers. Molecular orientations were visualized with a gypsum first-order red retardation plate inserted at 45° between the polarizer and analyzer [33 (link)]. Images were captured with a Lumenera Infinity 3 digital camera (Ottawa, ON, Canada). Freeze-dried samples were mounted onto aluminum stubs via adherence to double-sided copper tape. All of the samples were then sputter coated with amorphous carbon before analysis with an FEI Nova 430, operated at 15 kV and a spot size of 4.
Ultrathin sections were imaged with a Hitachi 760 TEM (Hitachi High-Technologies America, Schaumburg, IL, USA) equipped with a Macrofire monochrome progressive scan CCD camera (Optronics, Goleta, CA, USA) and AMT image capture software version 600.335p (Advanced Microscopy Techniques, Danvers, MA, USA) at an accelerating voltage of 80 kV in bright field TEM (BF-TEM) mode. To help to visualize the crystallographic orientation of the hydroxyapatite crystals, ultrathin sections were imaged in BF/SAED mode using a JEOL 2010F (Tokyo, Japan) operating at 200 kV. AFM imaging was conducted on an Asylum MFP-3D (Santa Barbara, CA, USA) operating in AC tapping mode with a nominal scan rate of 1 Hz.

Pseudo-nitzschia cultures were examined using a Nikon Eclipse TS100 inverted light microscope (LM) equipped with a Lumenera Infinity 3 digital camera (Ottawa, Canada) to a maximum magnification of ×400. For TEM examination, cultures were preserved in Lugol’s iodine prior to cleaning using the method of Hasle and Fryxell [40 ]. Once cleaned, 3 μl of each strain was placed on formvar-coated copper grids and loaded into a FEI Tecnai T20 TEM (LaB6), operated at a high tension of 120 kV and equipped with a Gatan 894 CCD 2k × 2k camera. Images for cell shape were obtained using LM, while all other frustule characteristics and morphometrics were obtained from TEM images, quantified using Image J1 (https://imagej.net/ImageJ1).

Hypothalamus sections of 4 μm thickness were prepared from paraffin-embedded specimens. To identify newborn neurons (DCX⁺), we conducted dewaxing, antigen retrieval, antibody incubation, staining, and sealing. DCX antigen was retrieved using sodium citrate (pH 6∙0). Sections were incubated 14-16h at 4 °C with the primary antibody anti-DCX (1:200; Cat# ab18723; Abcam). The sections were then incubated with goat anti-rabbit IgG (Cat# A16101, ThermoFisher Scientific, USA). Immunoreactive cells were visualized and captured using a fluorescence microscope (BX53, Olympus, Tokyo, Japan) and digital camera (INFINITY3, Lumenera, Ottawa, Canada) at 200 × magnification. ImageJ software v1.53e (National Institutes of Health, Bethesda, MD, USA) was used for analysis. n = 2 slices from 2 mice/group.