K3461

After perfusion and fixation, free-floating tissue sections were treated with peroxidase-blocking solution (S2023, Dako, Glostrup, Denmark) and incubated in protein-blocking solution (X0909, Dako). The sections were incubated with a primary antibody against STMN1 (1:200, ab52630; Abcam, Cambridge, UK) and biotinylated secondary antibodies (1:200, E0466; Dako). After washing with phosphate buffered saline, sections were placed in streptavidin-horseradish peroxidase solution (1:200, P0397; Dako) and incubated with the chromogen AEC (3-amino-9-ethylcarbazole, K3461, Dako).

The synovial membrane samples from body donors of the Friedrich-Alexander University Erlangen-Nuremberg which are embedded in paraffin were deparaffinized by incubating the slides in xylene for 10 min. Subsequently, the samples were incubated in a second xylene solution for 20 min before continuing the deparaffinization with ethanol (100%/100%/96%/80%/70%). After washing the slides twice with distilled water, they were incubated in 3% H2O2 for 10 min at room temperature. The slides were then washed three times with distilled water before being heated in citrate buffer (pH 6) for 10 min and then cooled to room temperature for at least 1 h. After a 10 min incubation with trypsin, the slides were washed with TBS-T and treated with normal goat serum for 20 min at room temperature. An avidin/biotin blocking kit (BioLegend, SIG-31126) was used for 10 min each before the primary antibody (GSN, Santa Cruz, 48749, 1:50) was added overnight at 4 °C. The next day, the slides were washed in TBS-T before the secondary antibody (DAKO, 86048, 1:200) was added for 1 h at room temperature. After incubation, the slides were washed three times and treated directly with an ABC kit (Vector Laboratories, PK-6100) for 1 h at room temperature before staining with an AEC solution (DAKO, K3461). The staining was stopped with distilled water and counterstained with hemalum before the slides were covered.