Rabbit anti tnfr2

Tissue blocks containing infarcted human brain tissue were imbedded in paraffin and 2 μm thick, serial sections were cut on a microtome, deparaffinized, blocked in 1.5% H₂O₂ in Tris-buffered saline (TBS) and demasked in T-EG buffer (10 mM Tris + 0.5 mM EGTA, pH 9.0), whereafter they were loaded onto a Dako autostainer (Dako, Denmark)61 (link). Sections were stained using the following primary antibodies: rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1,000, Wako), mouse anti-CD45 (1:200, Dako), mouse anti-CD68 (1:100, Dako), rabbit anti-GFAP (glial fibrillary acidic protein, 1:2,000, Dako), rabbit anti-TNF (1:100, Endogen)4 (link)23 (link)27 (link), rabbit anti-TNFR1 (1:50 (H-271), Santa Cruz), and rabbit anti-TNFR2 (1:50, Sigma-Aldrich). Secondary antibodies included EnVision + System horseradish peroxidase (HRP)-labelled Polymer (Dako) for CD45, CD68 and GFAP, Advance HRP (Dako) for TNF and TNFR1, and PowerVision + Poly-HRP IHC detection system (AH Diagnostics) for TNFR2. Omission of primary antibody and comparable concentrations of rabbit IgG were used to control for unspecific binding in the immunohistochemical protocols for TNF, TNFR1 and TNFR2 and these sections were devoid of staining. In addition, parallel sections were stained for hematoxylin and eosin (HE) using standard protocols at the Department of Pathology, Odense University Hospital.

Immunohistochemical staining was performed using the Dako autostainer platform (Dako, Denmark) as previously described [24 (link)]. Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems). The antigen-antibody complex was visualized using EnVision+System horse-radish peroxidase-labelled Polymer (Dako), PowerVision+Poly-HRP IHC (AH Diagnostics), or CSAII (Dako) detection systems.