Methods
Patients
15 AITL and 8 Hodgkin patients were included in this study. Informed consent was obtained from all subjects. The samples were retrieved from the files of the Institute for Hematopathology, Hamburg, Germany. Histological diagnoses were established according to the WHO classification [26, 27]. This study was conducted in accordance with the “Ethical Principles for Medical Research Involving Human Subjects” and approved by the Institutional Review Board of the Institute for Hematopathology, Hamburg, Germany.
Tissue probe stainings
Tissue probes were fixed in buffered 4% formaldehyde and routinely embedded in paraffin. Paraffin tissue sections (1 µm thick) were deparaffinized with xylene and rehydrated with graded ethanols. After dehydration and clearing in xylene, the sections were covered with permanent mounting medium and observed on an upright microscope (Zeiss Axio Imager.Z1) equipped with microscope cameras Zeiss Axio Cam.
Immunohistochemistry
Deparaffinized sections were subjected to antigen retrieval by heating in a steamer with sodium citrate buffer, pH 6.0, at 95°C x 30 min. Blocking the endogenous Fc receptors prior to incubation with primary antibodies was omitted [28]. After antigen retrieval and quenching endogenous peroxidase, sections were immunoreacted with primary antibodies (Supplementary Table 1). Immunohistochemical visualization of bound primary antibodies was performed either with Ventana Slide Stainer or manually according to the standard protocol [29, 30]. For manually performed immunostaining, primary antibodies were applied in concentration from 1 to 5 µg/ml.
For brightfield microscopy, bound primary antibodies were detected with AmpliStain™ Horseradish Peroxidase (HRP) conjugates (SDT GmbH, Baesweiler, Germany) according to the manufacturers’ instructions [31] and visualized using 3,3’-diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA, USA). The nuclei were counterstained with hematoxylin.
For fluorescence detection, bound primary antibodies were visualized using secondary antibodies (purchased from Dianova, Hamburg, Germany, and Molecular Probes, Darmstadt, Germany) conjugated with Cy3, Alexa Fluor-488 or Alexa Fluor-647. Nuclei were counterstained with DAPI (5 µg/ml in PBS) for 15 s, and the sections were then mounted using VectaShield (Vector Laboratories, Burlingame, USA). The list of secondary antibodies and other reagents used in this study is presented in the Supplementary Table 2.
Controls
Control incubations were: omission of primary antibodies or substitution of primary antibodies by the same IgG species (Dianova, Hamburg, Germany) at the same final concentration as the primary antibodies. The exclusion of either the primary or the secondary antibody from the immunohistochemical reaction, substitution of primary antibodies with the corresponding IgG at the same final concentration resulted in lack of immunostaining. The TSA step alone did not contribute to any specific immunostaining that might have influenced the analysis.