Iba 1

Brains were removed and dissected, post‐fixed in 4% paraformaldehyde (PFA) for 48 hr, and transferred to 20% sucrose in 0.1 M phosphate buffer (PB) for at least 16 h. Serial sections of the entire brain were cut at 25 μm thickness on a freezing cryostat (Leica Model: CM 3050 S). After blocking with 4% bovine serum albumin (Sigma‐Aldrich, St. Louis, MO, USA) and 0.3% Triton X‐100 in 0.1 M PB, sections were incubated with antibodies against ionized calcium‐binding adaptor molecule (polyclonal, IBA1; 1:100; Abcam, Cambridge, MA, USA) and kept at 4°C overnight. Control sections were incubated without the primary antibody. Sections were washed three times with 0.1 M PB and incubated in Alexa Fluor 488 goat anti‐mouse IgG (1:500, Thermo Fisher Scientific, Waltham, MA, USA) for 60 min at room temperature. Sections were mounted on slides and cover‐slipped. Confocal analysis was performed using a Nikon D‐ECLIPSE 80i microscope (Nikon Instruments, Inc., Tokyo, Japan) and the EZ‐C1 3.90 software (Nikon, Tokyo, Japan). The optical density of IBA1 immunoreactivity was quantified in three consecutive brain sections with a visualized anterior commissure in each animal, as previously reported.²⁵ Five photomicrographs were taken along the perilesioned region per brain slice.

To evaluate the effect of imipramine after TBI, we performed immunofluorescence 24 h after TBI [68 (link)]. Each sham and TBI group comprised 4 and 6 rats, respectively. We washed the brain tissues with 0.01% PBS 3 times for 10 min. Next, we soaked the brain tissues for 15 min in 1.2% hydrogen peroxide at room temperature to block intracellular peroxidase. We washed the brain tissues again for 10 min 3 times with 0.01% PBS. Next, we immersed the tissues in PBS solution containing Triton X-100 with the primary antibodies and kept overnight. In this study, we used 4-HNE (diluted 1:500; Alpha Diagnostic Intl. Inc., San Antonio, TX, USA), Iba1 (diluted 1:500; Abcam, Cambridge, UK), GFAP (diluted 1:1000, Abcam, Cambridge, UK), and MAP2 (diluted 1:200; Abcam) primary antibodies. Next, we washed the tissues with 0.01% PBS, after which we stained the secondary antibodies, 4-HNE and GFAP, with Alexa-Fluor-594 conjugated antibody, and we stained MAP2 and Iba1 with Alexa-Fluor-488 conjugated antibody (diluted 1:250, Invitrogen, Grand Island, NY, USA) with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, Grand Island, NY, USA) fluorescence staining for 2 h. Next, we mounted the stained brains with DPX, which we observed with a fluorescence microscope. Subsequently, we analyzed the results with Image J to measure the oxidative stress, microtubules, astrocytes, and microglia.

Mice were trans-cardiacally perfused with 4% paraformaldehyde. Their brains and colons were post-fixed with 4% paraformaldehyde for 4 h, cytoprotected in 30% sucrose solution, freezed, cut using a cryostat (Leica, Nussloch, Germany), and immunostained according to the method of Jang et al. (11 (link)). Briefly, the sections were washed with phosphate buffered saline, blocked with normal serum, incubated with antibodies for Iba1 (1:200, Abcam), LPS (1:200, Millipore), NF-κB (1:100, Cell Signaling), CD11b (1:200, Abcam), CD11c (1:200, Abcam), and/or NeuN (1:200, Millipore) overnight, and treated with the secondary antibodies for 2 h. Secondary antibodies conjugated with with Alexa Fluor 488 (1:200, Invitrogen) or Alexa Fluor 594 (1:200, Invitrogen) were then treated to visualize. Nuclei were stained with 4′,6-diamidino-2-phenylindole, dilactate (DAPI, Sigma). Immunostained tissue slices were scanned with a confocal laser microscope.

A double immunofluorescene procedure using NLRP3 (1:100, Santa Cruz Biotechnology; Santa Cruz, CA), CD31 (BD Biosciences; San Diego, CA), MAP2 (1:100, Merck/Millipore; Jaffrey, NH), GFAP (1:100, Merck/Millipore), and Iba‐1 (1:100, Abcam; Cambridge, MA) was performed as described previously. Photographs were taken with a confocal microscope (Leica; Solms, Germany) for further analysis.

Brains from time‐culled or terminally ill mice were fixed in 10 % buffered formal saline. Brains were dissected on the level of the striatum, the anterior hippocampus/thalamus and the midbrain with the rostral end of the cerebellum. Each of the slices was processed separately and embedded in paraffin wax. Serial coronal sections from the most caudal block, corresponding to the brainstem and the cerebellum, were cut at a nominal thickness of 3 μm, with five consecutive serial sections, followed by a 50 μm gap between levels, to allow a reproducible and consistent analysis of all brainstem regions. Brain sections were stained with haematoxylin‐eosin and prepared for immunohistochemical staining. Antibodies or antisera against the following antigens were used: PrP (ICSM35, D‐Gen London UK), glial fibrillary acidic protein (GFAP) (DAKO Z0334 Ely, UK), β‐galactosidase (Abcam Ab616 Cambridge, UK), ionized calcium‐binding adapter molecule 1 (Iba‐1) (Abcam 5076) and Neurokinin 1 (NK1) receptor (Pierce PA1‐32229 Hemel Hempstead, UK). All immunostainings were carried out on the automated Ventana Benchmark XT or Discovery XT (Roche Burgess Hill, UK) staining instrument, using biotinylated secondary antibodies and a horseradish peroxidase‐conjugated streptavidin complex and diaminobenzidine as a chromogen.