Reichert ultracut e ultramicrotome

One, three and five-day-old cyprids (each samples, n = 4) were relaxed with 8% magnesium chloride51 (link)52 53 (link) for 1 h and washed thoroughly with 0.45 μm filtered 33 ppt sea water. The cyprids were then fixed with modified Karnovsky (2% paraformaldehyde and 2.5% double-distilled glutaraldehyde buffered with 0.1 M sodium cacodylate containing 3% sucrose, pH 7.4) overnight at 4 °C. The specimens were washed with cacodylate buffer, post-fixed with 1% aqueous osmium tetroxide, OsO₄, washed again with cacodylate buffer, dehydrated through a graded series of ethanol, infiltrated and embedded in Agar epoxy Resin 100 (Agar Scientific). The embedded specimens were polymerised for 48 h at 60 °C. Semithin sections were cut with glass knives on a Reichert Ultracut E ultramicrotome (Reichert-Jung) to locate the target structures. The semithin sections were stained with toluidine blue and examined under Leica DMRB microscope equipped with the Leica QWin Plus software (Leica, Wetzlar,Germany). Serial ultrathin sections (60–90 nm) were cut with diamond knives (Diatome, Switzerland). The ultrathin sections were collected on bare 75, 100 and 150-mesh copper grids, double stained with uranyl acetate (for 8 min) and lead citrate54 (link) (for 5 min) and examined with FEI CM100 and Hitachi ST7700 transmission electron microscopes operating at 80 and 100 kV.

For transmission electron microscopy (TEM) analysis, 1 mpf WT (n= 3), tmem38b^-/- (n=3) and tmem38b^{Δ120-7/Δ120-7} (n=3) were fixed for 24 h at RT in 1.5% v/v PFA (Sigma Aldrich), 1.5% v/v glutaraldehyde (Sigma Aldrich), 0.1 M sodium cacodylate buffer (pH 7.4) and 0.001% w/v CaCl₂. The samples were decalcified in 0.1 M EDTA for 14 days at 4°C. Samples were rinsed in 0.1 M sodium cacodylate buffer containing 10% sucrose and post fixed for 2 h using 1% w/v OsO₄ in 0.1 M sodium cacodylate buffer at pH 7.4. Subsequently, zebrafish were infiltrated with low-viscosity epoxyembedding medium. Ultra-thin (70 nm) sections of the region of interest (vertebral endplate growth zone) were cut using a Reichert Ultracut E ultramicrotome (Reichert-Jung) with a diamond knife (Diatome Ltd.) and mounted on formvar-coated single slot copper grids. The sections were stained with uranyl acetate and lead citrate and viewed with a Jeol JEM-1010 (Jeol Ltd) TEM operating at 60 kV. Microphotographs were taken with a Veleta camera (Emsis, Germany) (30 (link)). TEM images were used to detect the endoplasmic reticulum cisternae enlargement. The area of ER cisternae in WT, tmem38b^-/- and tmem38b^{Δ120-7/Δ120-7} was measured using LAS v4.13 software (Leica).