Lsm 800 meta microscope

For immunofluorescence staining of PLA₂R1 and THSD7A 1 μm paraffin sections of kidney or lung tissue were deparaffinized and rehydrated with water. Antigen retrieval was obtained by boiling in citrate buffer pH 6.1 (both 15 min at constant 120°C). Unspecific binding was blocked with 5% horse serum (Vector) in phosphate-buffered saline (PBS) for 30 min at RT prior to incubation at 4°C o/n with primary antibodies (PLA₂R1: Atlas, rabbit primary antibody, 1:100; THSD7A: Atlas, mouse primary antibody, 1:100) in blocking buffer. Staining was visualized with fluorochrome-conjugated secondary antibodies (all affinity-purified from Jackson Immunoresearch Laboratories, 1:200) for 10 min RT in 2.5% horse serum in PBS. Nuclei were counterstained with DRAQ5 (Molecular Probes, 1:4,000). All negative controls were performed by omitting primary antibodies. Staining was evaluated with a confocal LSM 800 meta microscope using the LSM software (all Zeiss).

Tissue sections from the intestine were incubated overnight at 4 °C with primary antibodies, followed by incubation with Alexa Fluor‐labeled secondary antibodies for 1 h at room temperature. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich) and slides were washed with PBS, dried, and mounted using Antifade Mounting Medium (Invitrogen). For immunohistochemistry, formalin‐fixed, paraffin‐embedded sections (5 mm) were deparaffinized in xylene and in decreasing concentrations of alcohols, followed by antigen retrieval, quenching with 0.3% H₂O₂, and blocking with 3% BSA in PBS containing 0.1% Triton X‐100 for 60 min. Sections were then incubated overnight at 4 °C with the indicated antibody, followed by addition of a secondary horseradish peroxidase‐conjugated antibody (Invitrogen, 1:200) for 1 h and diaminobenzidine (DAB) chromogen according to the manufacturer's instructions. Finally, slides were counterstained with Mayer's haematoxylin (abs9214, Absin), dehydrated, and mounted with neutral balsam. Slides were visualized and captured using the Zeiss LSM 800 META microscope, and images were processed using ImageJ or Photoshop software. All images shown here were representatives of at least three randomly selected views.