DOI: 10.18150/z04vgn
title: The effect of nickel on the midgut of the freshwater shrimp Neocaridina davidi. Analyses: mitochondria (quantitative and qualitative) and HSP70, MnSOD proteins

description: This dataset contains the results of a study on the effect of nickel on the midgut of the shrimp Neocaridina davidi.During the experiment, the shrimp were divided into experimental groups, exposed to nickel, and returned to clean water to verify whether the changes were reversible. The animals were divided into the following experimental groups: CT – control – the animals stayed in clean water throughout the experiment;Ni 1:0 and Ni 2:0 – shrimps bred in the aquarium water with the addition of Ni for 1 and 2 weeks respectively;Ni 1:1 and Ni 1:2 – shrimps after a week’s cultivation in the aquarium water with the addition of Ni and returned to clean water and cultured for 1 and 2 weeks respectively;Ni 2:1 and Ni 2:2 – shrimps after 2 weeks of cultivation in the aquarium water with the addition of Ni and returned to clean water for 1 and 2 weeks respectively.The changes in the midgut, consisting of the intestine and the hepatopancreas, were analyzed.Attached are 6 zip files:1) Western blot - HSP70 and mtHSP70 (picture from the analysis for HSP70 and mtHSP proteins)2) Confocal microscopy - JC1- detection of depolarized mitochondria in cells (pictures of the intestine and hepatopancreas)3) Fluorescence microscopy: Hsp70 (pictures of the intestine and hepatopancreas)4) Fluorescence microscopy: MnSOD (pictures of the intestine and hepatopancreas)5) Flow cytometry - MitoPotential (the spreadsheet)6) Luminometry (the spreadsheet)Western blot - HSP70 and mtHSP70 -Description: Western blot analysis: Denatured samples (water bath, 5 min, 95 °C) of identical amounts of protein (25 µg) were loaded and separated by 10% SDS-PAGE (30 min at 90 V, then 1 h at 120 V) and then transferred to nitrocellulose membrane (Optitran BA-S 85, Whatman) with Mini Transfer-Blot (BIO-RAD) (2 h at 150 V, 300 mA). Next, the membranes were blocked (3% bovine serum albumin (BSA) in Tris-buffered saline (TBS), for 1 h, at room temperature (RT)). Blots were incubated with specific primary antibodies: HSP70 Monoclonal Antibody 5A5 (Invitrogen by Thermo Fisher Scientific) and mtHSP70 Monoclonal Antibody JG1 (Invitrogen by Thermo Fisher Scientific) (overnight, at 4 °C, with continuous shaking). After incubation, the membranes were washed four times for 5 min in TBS with 0.1% Tween-20 (TBST) and then incubated with a secondary antibody: goat anti-mouse IgG (pAB) AP conjugate (1 h, at RT, continuously shaking). The antibodies were diluted following the manufacturer’s instructions, in 1% BSA in TBS. After washing (4 × 5 min in TBST), the antibody complex was visualized using BCIP/NBT Solution (BioShop), washed again in distilled water, dried, and scanned. Colored lines indicate the size marker.Confocal microscopy - JC1- detection of depolarized mitochondria in cells -Confocal Microscope: Olympus FluoView FV1000, Magnification=60, Staining=JC1 + DAPI,Description: JC-1 differentiates cells with high mitochondrial potential (orange fluorescence; polarized mitochondria) and low mitochondrial potential (green fluorescence; depolarized mitochondria), nuclei are blue.Fluorescence microscopy: Hsp70Fluorescence Microscope Olympus BX63Description: For fluorescence microscopy analyses, tissues were fixed in 4% formalin, rinsed several times with PBS (phosphate buffer saline), and dehydrated in an ethanol series of increasing concentration (30%, 50%, 70%, 90%, 96%, and 100%). Then, the tissues were infused and embedded in Steedman's wax. The blocks were cut into 4 μm thick sections on a rotary microtome. The preparations prepared in this way were deparaffinized in an ethanol series of decreasing concentration (100%, 90%, 70%, 50%, 30%) and TBS, then rinsed in TBS + triton and 0.1% BSA and incubated in the primary antibody HSP70 (HSP70 Monoclonal Antibody 5A5, Invitrogen) (overnight, RT, in the dark). The slides were washed in TBS and incubated in a secondary antibody (goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488, Invitrogen) (2 h, RT, in the dark), rinsed in TBS, stained with DAPI (30 min, RT, in the dark), rinsed again in TBS, then the slides were mounted. The slides were analyzed in an OLYMPUS BX63 fluorescence microscope. Nuclei are blue, and Hsp70 proteins are green.Fluorescence microscopy: MnSODFluorescence Microscope Olympus BX63Description: For fluorescence microscopy analyses, tissues were fixed in 4% formalin, rinsed several times with PBS (phosphate buffer saline), and dehydrated in an ethanol series of increasing concentration (30%, 50%, 70%, 90%, 96%, and 100%). Then, the tissues were infused and embedded in Steedman's wax. The blocks were cut into 4 μm thick sections on a rotary microtome. The preparations prepared in this way were deparaffinized in an ethanol series of decreasing concentration (100%, 90%, 70%, 50%, 30%) and TBS, then rinsed in TBS + triton and 0.1% BSA and incubated in the primary antibody for MnSOD (anti-MnSOD rabbit polyclonal antibody, Stressgen) (overnight, RT, in the dark). The slides were washed in TBS and incubated in a secondary antibody (goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488, Invitrogen) (2 h, RT, in the dark), rinsed in TBS, stained with DAPI (30 min, RT, in the dark), rinsed again in TBS, then the slides were mounted. The slides were analyzed in an OLYMPUS BX63 fluorescence microscope. Nuclei are blue, and MnSOD is green.Flow cytometry - MitoPotentialFlow cytometer=Muse® Cell AnalyzerTest= MUSE MitoPotential KitDescription = Muse MitoPotential Kit is used in the quantitative analysis of cells with polarized/depolarized mitochondria.LuminometryDevice = Tecan infinite M200 with luminometric attachmentTests = ApoSENSOR ATP Cell Viability Bioluminescence Assay Kit (BioVision, № K254) and the ApoSENSOR ADP/ATP Ratio Bioluminescence Assay Kit (BioVision, № K255)Description = To assess ATP concentration and the ADP/ATP ratio, the following kits were used: the ApoSENSOR ATP Cell Viability Bioluminescence Assay Kit (BioVision, № K254) and the ApoSENSOR ADP/ATP Ratio Bioluminescence Assay Kit (BioVision, № K255). ATP concentration was determined based on the oxidative decarboxylation reaction of luciferin, catalyzed by luciferase in the presence of high-energy ATP and magnesium ions. The light intensity was measured at a wavelength of 562 nm. The files were converted to open files to facilitate access and subsequent use of the data (.ods and .jpg).