Veleta camera

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).

Hydrogels were visualized in transmission electron microscopy (TEM) using two approaches: negative staining of native gels and ultrathin sections. Copper TEM grids covered with formvar film preliminary stabilized with carbon evaporation were used for the sample adsorption.
To prepare negatively stained samples, semifluid hydrogels were adsorbed on a TEM grid for 1 min. In the case of more rigid hydrogels, a piece of about 2 mm³ was mixed with 2 μL of distilled water using a needle. Then, a grid was placed onto gel for 1 min, after that visible pieces of gel were gently removed by a needle. The grids with adsorbed samples of hydrogels were contrasted for 10 s on a drop of 2% phosphotungstic acid (pH 0.5). At least 5–6 grid-fields were examined for each sample in TEM.
To prepare ultrathin sections, hydrogels fixed in 4% paraformaldehyde were postfixed in 1% OsO₄, routinely dehydrated in ethanol and acetone and embedded in epon-araldit mixture. Ultrathin sections were prepared on a Leica EM UC7 ultratome (Leica Microsystems, Wein, Austria) and routinely contrasted with uranyl acetate and led citrate. All grids were examined in JEM 1400 TEM (JEOL, Tokyo, Japan), digital images were collected with a Veleta camera (EMSIS, Muenster, Germany). At least 10 individual ultrathin sections of each sample were examined in TEM.