Ba 1100

Regarding the antigen retrieval, tissue sections were exposed to microwaves for 15 min at 600 W in 10 mM sodium citrate while H9c2 cells were treated with 0.2%, triton-X100/PBS (Sigma-Aldrich) for 10 min. Then the samples were incubated for 10 min in 3% H₂O₂/methanol in order to block endogenous peroxidases, treated for 20 min at 37 °C with PBS-blocking solution containing 1% normal goat serum (NGS, Vector, Burlingame, CA, USA), 0.1% bovine serum albumin (BSA, Sigma-Aldrich), 0.1% triton-X100 for blocking non-specific binding. and then incubated overnight at 4 °C with primary antibodies diluted in a 1%BSA/2%FBS/PBS solution (RαCx26-Ct 1:400, RαCx26-cl 1:200, RαCx43 1:200, RαCx32 1:200 and MαMyo 1:750). Detection was accomplished by sequential treatments with biotinylated anti-rabbit or anti-mouse immunoglobulins (1:200, BA-1100 or BA-2000, Vector), streptavidin-peroxidase complex (Vector) and 3.3’diaminobenzidine tetrahydro-chloride (DAB Dako Italia SRL, Milan, Italy). The specificity of secondary antibodies was obtained by performing the experiments omitting the primary antibodies. Sections were then counterstained with Harris’ hematoxylin (Fluka, Buchs, Switzerland) and examined by means of a Leica DMRB light microscope at 20x or 40x magnification; representative images were captured by DFC480 digital camera (Leica Microsystem, Cambridge, UK).

Animals were perfused with saline, followed by 4% paraformaldehyde (PFA) solution and brains were dissected out and stored in 4% PFA. Brains were sectioned on a vibratome (Leica VT1000 S, U.S.A.) at 40-μm thickness. Free-floating sections spanning the PVN of the hypothalamus (−0.58 to −1.22 mm from Bregma, five to six sections per animal) and PFC (2.46–1.34 mm from Bregma, five to six sections per animal) were subjected to processing for c-Fos immunohistochemistry. Briefly, sections were blocked in 10% horse serum, followed by overnight incubation with rabbit-anti-c-Fos antibody (1:500, Catalog number CST-2250S, Cell Signaling Technology). Sections were washed and incubated with a biotinylated horse-anti-rabbit IgG secondary antibody (1:500, Catalog number BA-1100, Vector Laboratories). Signal amplification and visualization involved exposure to an avidin–biotin complex solution (ABC solution, Vector Laboratories) followed by 3,3′-Diaminobenzidine (DAB) substrate (Sigma, U.S.A.). Sections were mounted and visualized at 200× magnification (Zeiss Axioskop, Germany). c-Fos positive cells were counted per section by an experimenter blinded to the treatment conditions.

Human colon cancer TMA from USBioLab (Maryland, USA) was deparaffinized using xylene and rehydrated using graded ethanol (100%, 95%, 70%, 50%) and distilled water. Antigen retrieval was performed using pH 6 citrate buffer in pressurized heater for 16 min. After cooling, samples were permeated using 2% Triton X-100/TBS for 30 min. Endogenous peroxidase, avidin and biotin were blocked using 3% hydrogen peroxide, avidin and biotin blocking solutions in 2% normal horse serum (NHS) (Vector Labs #SP-2001, #S-2000). TMAs were incubated with anti-NOD1 (1:200) or NHS for vehicle control at 4 °C overnight. The next day, after washing with 0.05% TBST, biotinylated secondary antibody (Vector Labs #BA-1100), streptavidin-HRP, DAB chromogen (Vector Labs VECTASTAIN Elite ABC kit #PK-6100, ImmPACT DAB Peroxidase substrate #SK-4105) and Hematoxylin (Sigma-Aldrich) were sequentially added before slide dehydration in graded ethanol and xylene and coverslip with Permount mounting medium (Fischer Scientific). After drying, the specimens were examined twice by a Royal College-certified pathologist (Dr. R. McClure) who is blinded and have no prior knowledge of the study. Each core staining intensity was scored as “3-Strong-staining”, “2-Some-staining” or “1-No-staining”.