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.