Clone l26

In parallel, the spectral library was created for multispectral image analysis visualization extraction. Using an antibody with higher amount of positive cells in tonsils as CD20 antibody (B-cell marker; clone L26, dilution 1:100; Dako), the eight fluorophore tyramides were stained using similar conditions as for the single IF protocol without DAPI while trying to maintain similar range of expression (Supplementary Fig. 2).

Mouse spleens and livers were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections of 10 μm thickness were deparaffinized and antigens were retrieved using citrate-based buffer (pH 6) for 10 min (H3300, Vector Laboratories). Slides were incubated in blocking solution (3% bovine serum albumin + tris-buffered saline with Tween 20) for 1 h and incubated overnight with mouse anti-human CD3 (Dako, Clone F7.2.38, #M725401-2, 1:100) and mouse anti-human CD20cy (Dako, Clone L26, #IR604, 1:50). Slides were then incubated with an HRP-conjugated secondary antibody (Dako EnVision+ Dual Link System-HRP, Dako, #K4063). The slides were developed with diaminobenzidine (ImmPACT DAB Peroxidase (HRP) Substrate, Vector Laboratories, #SK-4105).

Human kidney transplant biopsies were fixed in 4% neutral buffered formaldehyde and embedded in paraffin. Two μm sections were stained for routine histochemical diagnostics (H&E, PAS, Jones Methenamine) according to standard protocols, immunohistochemical stains were performed for CD20 (Dako, clone L26, 1:500 after heat pretreatment with EDTA at pH8.4 for 16 minutes) on an automated platform (Ventana Benchmark Ultra). Normal biopsies can contain single B-cell infiltrates without any clinical relevance. Therefore, we developed a cutoff of >30 CD20+ cells per hpf for definition of relevant “B-cell rich” infiltrates. For the evaluation we used a visual analog scale that we have developed ourselves for standardized evaluation of the samples Figure S3).

In brief, sinonasal tissues were dehydrated and embedded in the paraffin and sectioned at 3 µm diameters. After rehydration and blocking of the endogenous peroxidase activity with 3% H₂O₂/methanol, the sections were washed with Tris-buffered saline (TBS) and blocked (with PBS, pH 7.4, containing 2% bovine serum albumin (Sigma-Aldrich, Darmstadt, Germany), 0.1% Triton X-100, and 0.1% sodium azide) at room temperature (RT) to reduce nonspecific bindings for 30 min to hinder nonspecific binding [21 (link)]. Then, the sections were incubated with an appropriate concentration of the antibodies for 1 h at RT. The details of which are as follows: primary antibodies, including polyclonal rabbit anti-human IgA antibody (at 1:100 dilution; Abcam, Cambridge, MA, USA), anti-human IgA1 antibody (at 1:200 dilution; ab193187), anti-human IgA2 antibody (1:100; ab193169, Abcam), monoclonal mouse anti-human CD20 (at 1:200 dilution; clone L26, Dako, Glostrup, Denmark), anti-human CD138 (1:100; Clone M115) were applied. After 2 h incubation, the slides were washed with TBS for 10 min and again incubated for 45 min at 30 °C with EnVision™ (Dako). The samples were coun-terstained with Mayer’s hematoxylin stain and mounted in Faramount Mounting Medium (Dako), before microscopic examination.