Image data of co-localization of IgG and HEV ORF2 protein in a case of hepatitis E-associated kidney disease

Image data for a co-localization study of IgG with HEV ORF2 protein in a de novo immune complex-mediated glomerulonephritis (GN) case in a kidney transplant recipient with chronic hepatitis E (Leblond and Helmchen, et al. 2024). Immunofluorescence images are provided for 25 glomeruli at low magnification (20x, 0.227 micron/pixel) and for 16 glomeruli at high magnification (100x, 0.0454 micron/pixel). For each example glomeruli the green channel represents IgG antibody staining with FITC, and the magenta channel represent anti-HEV ORF2 staining using Alexa Fluor 546. Methods: Mouse monoclonal antibody clone 1E6 against the HEV ORF2 protein was incubated for 1h at a dilution of 1:125 followed by a mix of Alexa Fluor 546-conjugated goat anti-mouse antibody (Invitrogen BV, A11018) and FITC-conjugated Rabbit anti-Human IgG (Gamma chain, Diagnostic Biosystem, F008) for 1hat a dilution of 1:50. Following automated staining, the slides were hand -washed in distilled H2O. Tissue was covered with Vectashield® Antifade Mounting Medium with DAPI (VectorLaboratories, H-1200), covered with a coverslip and stored at 4°C until evaluation. Immunofluorescence images were acquired with an upright fluorescence microscope (AxioImager.Z2 controlled by ZEN Blue software; 89 North Photofluor LM-75 light source, and Axiocam 503 mono camera; Zeiss, Jena, Germany), equipped with the following objectives: 20x (NA 0.5, Plan-NEOFLUAR), 40x (NA 1.4 oil, Plan-APOCHROMAT), and 100x (NA 1.45 oil, Plan-APOCHROMAT) objectives. This setup provides an excellent spatial resolution (nominally about 200 nm lateral resolution in our study; pixel size was 45.4 nm for 100x objective). High resolution images were taken with the 100x objective using the ApoTome.2 module with deconvolution (grid 5 lp/mm; section thickness 0.7 µm). We used Vysis Abbott Chroma filter sets (Blue: excitation (ex) 335-383 nm, emission (em) 420-470; green: ex 481-507 nm; em 521-551 nm; red: ex 534-556 nm, em 574- 606 nm). Co-localization of IgG and HEV ORF2 staining was quantified using Fiji software (Schindelin et al., 2012) and the JACoP ImageJ plug-in.本数据集为肾移植术后慢性戊型肝炎患者新发免疫复合物介导性肾小球肾炎（GN）病例中，免疫球蛋白G（IgG）与戊型肝炎病毒（HEV）ORF2蛋白共定位研究的图像数据（Leblond与Helmchen等，2024）。共提供25个肾小球的低倍镜（20×，像素尺寸0.227 μm/像素）图像，以及16个肾小球的高倍镜（100×，像素尺寸0.0454 μm/像素）图像。每个目标肾小球的绿色通道代表异硫氰酸荧光素（FITC）标记的IgG抗体染色，品红色通道代表使用Alexa Fluor 546标记的抗HEV ORF2抗体染色。 实验方法： 针对HEV ORF2蛋白的小鼠单克隆抗体克隆1E6以1:125的稀释度孵育1小时，随后使用Alexa Fluor 546偶联山羊抗小鼠抗体（Invitrogen BV，货号A11018）与FITC偶联兔抗人IgG（γ链，Diagnostic Biosystem，货号F008）以1:50的稀释度混合孵育1小时。自动染色完成后，载玻片经手工蒸馏水冲洗。组织切片使用含4',6-二脒基-2-苯基吲哚（DAPI）的Vectashield®抗荧光淬灭封片剂（Vector Laboratories，货号H-1200）封片，加盖玻片后置于4℃保存直至成像评估。 免疫荧光图像采用正置荧光显微镜采集（AxioImager.Z2，由ZEN Blue软件控制；光源为89 North Photofluor LM-75，相机为Axiocam 503 mono；蔡司，德国耶拿），搭配以下物镜：20×（数值孔径NA 0.5，Plan-NEOFLUAR系列）、40×（NA 1.4 油镜，Plan-APOCHROMAT系列）及100×（NA 1.45 油镜，Plan-APOCHROMAT系列）。本研究的空间分辨率标称约200 nm横向分辨率；100×物镜对应的像素尺寸为45.4 nm。高分辨率图像采用100×物镜搭配ApoTome.2模块并经反卷积算法处理采集（光栅参数5 lp/mm，切片厚度0.7 μm）。所用滤色镜组为Vysis Abbott Chroma系列（蓝色通道：激发光335~383 nm，发射光420~470 nm；绿色通道：激发光481~507 nm，发射光521~551 nm；红色通道：激发光534~556 nm，发射光574~606 nm）。IgG与HEV ORF2染色的共定位情况通过Fiji软件（Schindelin等，2012）及JACoP ImageJ插件进行定量分析。