Tnfr2

Under anesthesia, mice were placed in a stereotaxic frame, and a 24-G stainless steel guide cannula was implanted 1 mm above the lateral cerebral ventricle and fixed in place with dental acrylic, as described previously [29 (link)]. After 1 day recovery, mice were anesthetized with pentobarbital and underwent 90 min right middle cerebral artery occlusion (MCAO) followed by reperfusion as described previously [9 (link)]. Anti-mouseTNFR1 (R&D Systems, Minneapolis, MN, USA) or TNFR2 (BioLegend, San Diego, CA, USA) monoclonal antibody (each in doses of 10, 50, and 100 ng/day) was given intracerebroventricularly (i.c.v.) through a pre-implanted guide cannula into the right lateral cerebral ventricles within 5 min after 90 min MCAO while animals were still anesthetized. Each mouse received daily i.c.v injections consecutively for 4 days of reperfusion following 90 min MCAO. As a control, purified hamster IgG alone was injected into the lateral ventricle of each mouse. Accurate placement of the injection or needle track was verified at the time of dissection. Mice were euthanized 4, 7, or 14 days post-ischemia.

TNF (Cat. No. 430201, BioLegend, San Diego, CA, USA), TNFR1, TNFR2, TGβ-1, and TIM3 (Cat. No. DY225, DY726, DY240, and DY2365 respectively; R&D Systems, Minneapolis, MN 55413, USA), and ADAM17 (Cat. No. SEB555Hu 96T, Cloud-Clone Corp., Katy, TX, USA) were evaluated. All the molecules were quantified by an ELISA assay in the sera, complying with the manufacturer’s instructions.

The frequency of immunological cellular subpopulations in pleural cells was analyzed by flow cytometry. Briefly, cells were stained for 30 min at 4 °C with following fluorochrome-conjugated mAb: CD11b (Clone M1/70) and TNFR2 (Clone TR75-89) (BioLegend, San Diego, CA, USA). Cells were incubated with antibodies and then washed with PBA (phosphate buffered saline containing 0.1% Sodium Azide and 1% Albumin bovine). We used a FACs CyAn flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and analyzed the data with the FlowJo (Tree Star, Ashland, OR, USA) software. We acquired 100,000 events per sample.