Detailed In Situ Hybridization Protocol for TNF-α Detection

In situ hybridization (ISH) was carried out as our previous description [17 (link)]. Briefly, 18-μm-thick frozen brain sections were fixed in 4% PFA for 15 min at room temperature. After equilibration in hybridization buffer (50% formamide, 5 × SSC, 40 mg/ml salmon sperm DNA), sections were hybridized with the Digoxigenin (DIG)-labeled TNF-α (NM_013693.2, 575-1607 bp) cRNA probes in hybridization buffer overnight at 60 °C. After washing and antigen blocking, the sections were incubated with alkaline phosphatase-conjugated anti-DIG antibody (Roche, Basel, Switzerland) at room temperature 2 h. For color development, 4-nitro blue tetrazolium chloride (NBT) (Roche Diagnostic Gmbh, Mannheim, Germany) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (Roche Diagnostic Gmbh, Mannheim, Germany) were incubated for 8 h at room temperature. Images of the stained sections were taken by Nikon digital camera system (DS-Fi1, Nikon Corp., Tokyo, Japan) in combination with microscopy (Nikon Eclipse 80i).