Mean SEM, *p 0

Mean SEM, *p 0.05 males (M) vs. response following PRHX TBI, not only for its hepatic and bloodstream levels but also for its expression in the injured brain. skim milk powder in PBS-Tween 20 (PBS-T) blocking buffer for 1 h at room temperature. Membranes were incubated with goat anti-SAA primary antibody (1:500; AF2948, R&D Systems, Minneapolis, MN) overnight at 4C and then with horseradish peroxidase-conjugated rabbit anti-goat secondary antibody (1:3000; Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature. Membranes were developed with Clarity Western ECL (Bio-Rad Laboratories, Hercules, CA). A ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA) was used for the Stain-Free and the chemiluminescence imaging and densitometry quantification was performed using the ImageLab 6.0.1 software (Bio-Rad Laboratories, Hercules, CA). Immunofluorescence analysis. A sliding microtome (Microm HM 430, Thermo Fisher Scientific, Waltham, MA) was used to section the brains at 20 m-thick in coronal orientation through the dorsal hippocampus. The brain sections were then cryoprotected in an antifreeze solution (30% glycerol + 30% ethylene glycol + 40% 0.01 M PBS) for storage at ?20C. Free-floating parallel brain sections were washed with PBS with L-Palmitoylcarnitine 0.5% Triton X-100 (PBS-T) 3 x 5 min and blocked with 5% normal horse serum (NHS, #S2000, Vector laboratories, Burlingame, CA) in PBS-T for 1 h. Brain sections were incubated at 4C overnight in PBS-T and 3% of NHS using the following primary antibodies: SAA, anti-rabbit TLR4 (1:200, sc-10741, Santa Cruz Biotechnology, Dallas, TX) for activated microglia/macrophages, anti-rat Ly-6B.2 alloantigen (1:100, MCA771GA, Bio-Rad Laboratories, Hercules, CA) for neutrophils, or anti-rat CD11b/c (OX-42, 1:200, Cat. 554862, BD Biosciences) is expressed on the surface of monocytes/macrophages, granulocytes, dendritic cells, and microglia in the brain. After incubation, brain sections were washed in PBS-T and incubated with the corresponding anti-rabbit Alexa Fluor 568-conjugated or anti-mouse Alexa Fluor 488-conjugated IgG secondary antibodies (all 1:1000, Thermo Fisher Scientific, Waltham, MA) for 2 h at room temperature. Sections were rinsed with PBS 3 x 5 min and incubated in PBS with DAPI solution (1:50,000, Sigma-Aldrich, St. Louis, MO) for counterstained nuclei. The sections were rinsed with distilled water and coverslipped with Fluoro-Gel with Tris Buffer mounting L-Palmitoylcarnitine medium (Electron Microscopy Sciences, Hatfield, PA). Brain sections for negative controls were incubated without the primary antibody (Supplementary Fig. 1). For quantitative analysis of immunolabeled sections, we implemented unbiased standardized sampling techniques to measure tissue areas corresponding to the injured cortex showing positive immunoreactivity as we previously described (Villapol et al. 2015; Villapol et al. 2014). To quantify the number of Ly6B.2, TLR4 and SAA-positive cells in the injured cortex, an average of four coronal sections from the lesion epicenter (?1.34 to ?2.30 mm from bregma) were counted and imaged for each animal, n= 5 mice per group. Within each brain region, positive cells were counted in each of 5 L-Palmitoylcarnitine cortical fields (x20 objetive, 151.894 mm2/field) around the impact area, as we have previously described (Villapol et al. 2017). All the investigators were binded for the experimental groups. Images were acquired on a Nikon motorized fluorescence microscope (Eclipse Ni-U, Melville, NY) with a pco.Edge Scomos camera (4.2LT USB3) and analyzed using NIS-Elements software. RNAscope? in situ hybridization combined with immunohistochemistry Coronal brain sections were L-Palmitoylcarnitine mounted on gelatin-coated glass slides (Superfrost Plus, Thermo Fisher Scientific) and stored at ?80C until use. Fluorescent in situ hybridization (FISH) was performed as per the manufacturers instructions using RNAscope? Technology 2.0 Red Fluorescent kit (Advanced Cell Diagnostics (ACD), Hayward, CA) as we previously described (Fe Lanfranco et al. 2017; Villapol et al. 2017). Brain tissue sections were dehydrated by 50%, 70%, and 100% ethanol gradually for 5 min; boiled during 10 min with pretreatment 2 solution (citrate buffer), then they were incubated with pretreatment 3 solution (protease buffer) for 30 min before hybridization. Sections were then incubated at 40C for 2 h with the following target probe for mouse: mRNA (Cat. No. 450191, ACD). In addition, the negative (Cat. No. 310043, ACD) and positive (Cat. No. 313911, ACD) control probes were applied and let hybridized for 2 h at 40C. The amplification steps were performed according to manufacturers directions. After FISH, slides were washed three times with PBS and blocking with PBST and 5% normal goat serum for 1 h. Immunofluorescence was performed using a mixture of primary antibodies incubated overnight at 4C; polyclonal anti-rabbit L-Palmitoylcarnitine Iba-1 (1:500, 019-9741, Wako Chemicals, Richmond, VA) for microglia/macrophages cells, monoclonal anti-mouse GFAP (1:500, AB5541, Millipore, IL) for astrocytes, and polyclonal anti-rat F4/80 (1:200, MABF152B, Sigma) for macrophage cells. Alexa Fluor 488-conjugated goat anti-rabbit, and anti-rat IgG (1:1,000, Invitrogen,.