Advance hrp link

For multiplex immunohistofluorescence, the Opal™ 4‐Color Manual Kit (PerkinElmer, Waltham, MA, USA) was used according to the manufacturer's instructions. Briefly, slides were deparaffinized and rehydrated and epitope retrieval was performed by microwave in AR6 buffer. Endogenous peroxidase activity and nonspecific binding were blocked. Incubation with primary antibodies (CD68; Santa Cruz, sc‐70763, 1 : 5000; LAPTM5; Abcam, ab108014, 1 : 100; RSK1 Santa Cruz, sc‐231, 1 : 5000), diluted in Tris/HCl (50 mm) pH 7.5 + 1% BSA, was performed overnight at 4 °C. After washing, secondary antibody Advance HRP Link (Dako^®‐K4068) was used. The fluorophore of the Opal working solution diluted 1 : 1000 in 1X amplification diluent was applied, and the slides were incubated for 10 min. Removal of the antibodies was performed with AR6 buffer in microwave, followed by a second round of primary and secondary antibodies. Nuclear staining was performed with Draq5 (Thermo Fisher, Waltham, MA, USA). Images were acquired by Leica SP5 confocal microscopy (Leica).

Formalin-fixed spleen samples were dehydrated and paraffin-embedded. Spleen samples were sectioned into 3-μm-thick pieces and placed on a salinized surface. Sections were deparaffinized with xylene and rehydrated with ethanol, activity of endogenous peroxidases was blocked for immunohistochemical staining, which was previously described by Smalinskiene and others [38 (link)]. Briefly, slides were incubated with primary anti-CD3 rabbit/mouse antibodies (DakoCytomation, Glostrup, Denmark), dilution 1:300 for 1 h. Unbound antibodies were washed after finishing incubation with primary antibody, the samples underwent sequential incubations with Advance HRP Link and Advantage HRP Enzyme reagents (Dako, Glostrup, Denmark) for 30 min. Staining was developed with liquid 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich Chemie GmBh, Steinheim, Germany) and Substrate Chromogen system (Dako, Glostrup, Denmark).

For histology, aggregates were fixed in 4% paraformaldehyde for 1 h, followed by dehydration in graded alcohols, paraffin embedding, sectioning to 4 μm, and staining with haematoxylin/eosin (H&E) or alcian blue (Sigma) as described previously [7 (link), 8 (link)]. For visualisation of ALP activity, a histochemical assay was performed according to the instructions of the supplier (Sigma).
Immunohistochemistry on alternate sections was performed as described previously [7 (link)]. Briefly, following the respective pre-treatments with pepsin (1 mg/mL), or chondroitinase ABC (Sigma; 5 U/mL), or trypsin (0.25%) sections were incubated overnight with the following primary antibodies: monoclonal anti-COL type II (Acris Antibodies GmbH, Hiddenhausen, Germany), anti-chondroitin-4-sulphate (CS4) (Millipore GmbH, Schwalbach, Germany) or anti-collagen type X (COL type X) (Calbiochem, Bad Soden, Germany). Immunohistodetection was performed by treatment with Advance™ HRP link and Advance™ HRP enzyme (Dako, Hamburg, Germany) followed by diaminobenzidine staining (DAB kit; Sigma), and slides were finally counterstained with hemalaun (Merck, Darmstadt, Germany). In addition, controls with non-immune Ig G (Sigma) instead of the primary antibodies were also performed.