Varistain gemini es automated slide stainer

Fin clipped larvae were fixed in 4% PFA at 4 °C overnight and kept in 70% ethanol. At least five embryos or larvae per genotype group were embedded in 1% agarose in 1X TAE buffer. A mould, specifically designed to align zebrafish larvae, was used to produce agarose blocks with identical distributed wells of the same depth. Agarose blocks were gradually dehydrated in an enclosed automated tissue processor (Shandon Excelsior ES, Thermo Scientific) and subsequently embedded in paraffin. The heads of paraffin-embedded larvae were sectioned on a HM 325 manual rotary microtome (Thermo Fisher Scientific) at a thickness of 5 μm. The specimens were stained with hematoxylin and eosin (H&E stain) using Varistain™ Gemini ES Automated Slide Stainer (Thermo Fisher Scientific) according to laboratory protocols. The resulting sections were imaged at ×20 magnification in a SPOT 5.1 software (SPOT Imaging) by a SPOT-RT3 camera mounted on a Leica microscope. Brightness of the images was adjusted for the white background.

Tissue sections (4 μm) were deparaffinized, rehydrated, washed and heated in 0.01 M citrate buffer (pH 6) at 125 °C for 5 min and at 90 °C for 10 min in a decloaking chamber (Biocare Medical). Sections were stained using the rabbit anti-PMCA2 ATPase polyclonal antibody (1:300; PA1-915 Thermo-Fisher Scientific) or β-casein monoclonal antibody (1:100; sc-53189, Santa Cruz), and the MACH-1 Universal HRP-Polymer Detection Kit (Biocare Medical) according to the manufacturer’s instructions. Nuclei were counterstained with hematoxylin using a Varistain Gemini ES Automated Slide Stainer (Thermo Fisher Scientific). The negative and positive controls were no primary antibody and cerebellar tissue, respectively. Stained tissue sections were scanned at 20× magnification using a ScanScope XT Digital Slide Scanner (Aperio), and evaluated by a blinded pathologist (LdS) using the following criteria: (1) positive: intense plasma membrane staining with or without cytoplasmic staining; (2) negative: cytoplasmic or no staining (since PMCA2 is a plasma membrane Ca²⁺-transporter).

Histological structure visualization of liver tissues were done by routine topographic staining with hematoxylin and eosin (H&E). The liver tissue slides were stained using an automatic stainer (Thermo Scientific, Varistain Gemini ES Automated Slide Stainer) following a standard protocol. First, 5 μm thick liver slices were deparaffinized and rehydrated by serial incubations with xylene and alcohol mixtures of different percentages. Then, the tissue was stained for 2 min in Mayer's Hematoxylin solution (O. Kindler &ORSAtec), differentiated in acid alcohol (1% HCl) for 1 min, washed in bluing reagent (Shandon) for 4 min, and incubated for 7 min in 0.2% EOSIN Y alcoholic solution (O. Kindler &ORSAtec). All steps were separated by 1 min washes in distilled water. Next, the liver tissue slices were dehydrated using a number of washes in alcohol and xylene mixtures of different percentages (70, 96, 100% ethanol for 30 s each and 3 changes of xylene, 1 min each). Finally, the slides were manually mounted with the xylene based mounting medium (Consul-Mount, Thermo Scientific Shandon) and coverslipped.