Cryo gel

For histological studies, the brains were isolated and fixed in 10% formalin solution at room temperature for 2 days and then placed in 15% sucrose solution (24–48 h) followed by 30% sucrose solution (24–48 h). The samples were then transferred to a Leica CM1520 freezing sliding cryostat (Leica, Germany) and gradually filled with cryogel (Leica, Germany). The brain was cut into 15-µm coronal sections, which were placed on slides and dried in the air for 24 h. They were stained according to a standard hematoxylin-eosin method (PanReac AppliChem, Germany). Next, the slices were dehydrated in alcohol solutions of increasing concentrations, purified in xylene, and embedded in mounting medium (Thermo Fisher Scientific, United States). The samples were examined using a Zeiss Primo Star light microscope (Zeiss, Germany) with an integrated Axio CamMRc camera (Zeiss, Germany).

Mouse brains were surgically removed from the skull and placed in a 10% formalin solution at room temperature for 24 h. Next, the brain samples were incubated in a 15% sucrose solution (24–48 h) and then stored in a 30% sucrose solution for 24–48 h. Each brain was then placed in a Leica CM1520 freezing sliding cryostat (Leica, Wetzlar, Germany), progressively filled with Cryogel (Leica, Wetzlar, Germany) and sliced into 10 µm coronal sections. Every fifth brain section was mounted on a glass slide, air-dried for 24 h, and stained with hematoxylin–eosin (PanReac AppliChem, Darmstadt, Germany). The brain sections were dehydrated in increasing concentrations of alcohol, purified in xylene, and embedded in mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). The resulting samples were examined with a Zeiss Primo Star light microscope (Zeiss, Oberkochen, Germany) with an integrated Axio CamMRc camera (Zeiss, Oberkochen, Germany).

Femora were isolated, fixed with 4% PFA (AppliChem) and 5 mM sucrose (Sigma-Aldrich), transferred into 10% sucrose in PBS and dehydrated using a graded sucrose series. Subsequently, the samples were embedded in Cryo-Gel (Leica Biosystems) and shock frozen in liquid nitrogen. Frozen samples were stored at −80 °C. Seven-micrometer-thick cryosections were generated using the CryoJane tape transfer system (Leica Biosystems) and probed with Alexa488-conjugated anti-GPIX antibody, to specifically label platelets and MKs, and Alexa647-conjugated anti-CD105 antibodies (3.33 mg ml⁻¹, 120402 (MJ7/18), Biolegend) to stain the endothelium. Nuclei were stained using DAPI (4,6-diamidino-2-phenylindole; 1 mg ml⁻¹, D1306, Invitrogen). Samples were visualized with a Leica TCS SP5 confocal microscope (Leica Microsystems).

Gut permeability was determined by fluorescein isothiocyanate dextran (FITC-dextran) assay, endotoxemia, and immunofluorescent detection of a tight junction protein (zonaoccludens-1; ZO-1) following previous publications^{33 (link)–35 (link)}. As such, FITC-dextran, a nonabsorbable molecule with 4.4 kDa molecular mass (Sigma-Aldrich, St. Louis, MO, USA) at 12.5 mg per mice was orally administered at 3 h before the detection of FITC-dextran in serum by Fluorospectrometer (NanoDrop 3300; Thermo Fisher Scientific, Wilmington, DE, USA). Serum endotoxin (LPS) was measured by HEK-Blue LPS Detection (InvivoGen, San Diego, CA, USA) and the data were recorded as 0 when LPS values were less than 0.01 EU/ml because of the limited lower range of the standard curve. Also, the cecum was used as a representative of the intestine to determine gut tight junction. Accordingly, cecum in Cryogel (Leica Biosystems, Richmond, IL, USA) were cut into 5 μm-thick frozen sections, fixed in acetone, blocked by blocking buffer, stained with a fluorescent antibody against ZO-1 and a green secondary antibody (Alexa Fluor 488) (Life Technologies, Carlsbad, CA, USA) before visualization and scoring by ZEISS LSM 800 (Carl Zeiss, Germany).

After being fixed with 4% Paraformaldehyde aqueous solution (Gibco/Thermo Fisher Scientific), samples were dehydrated with increasing gradient sucrose solutions and then frozen in Cryo-Gel (LeicaBiosystems, Richmond, IL, United States). Cryosectioning was done using the Leica CM1850 Cryostat (Mercedes Scientific, Lakewood Ranch, FL, United States) at 12 μm thickness. Senescence β-Galactosidase Staining Kit (BioVision, Milpitas, CA, United States) was used to assess β-galactosidase activity according to the manufacturer’s instructions.