Cm3050

Mouse brains were isolated from 3–6-month-old male mice without prior perfusion within 150 +/−30 s, mounted onto cryostat sockets (Leica CM3050S, Nussloch, Germany) and frozen in liquid nitrogen immediately. Subsequently, samples were stored in liquid nitrogen until further processing (max. 1 h). Each brain was completely processed the same day according to the workflow described below. Brains were cut in coronal orientation at a thickness of 8 µm using a cryostat (Leica CM3050S, Nussloch, Germany), starting from the onset of the hippocampal formation until the end. The Allen Mouse Brain Atlas (Allen Brain Institute) was used as a refs. ^{20 (link)–30 (link)}. laser dissection membrane slides (Molecular Machines and Industries GmbH, Eching, Germany) with 5–7 brain slices each were collected from each brain. All slides were subsequently used for tissue collection to avoid a collection bias alongside the rostro-caudal axis.

Histological analysis and immunohistochemistry staining were performed as previously described [38 (link)]. OCT-embedded common carotid arteries were cut systematically in serial 5-μm cross sections using a cryotome (Leica CM3050S, Leica Microsystems, Germany). Analysis was carried out in the injured left common carotid artery, whereas the contralateral served as a control. For morphometric analysis, sections were stained with hematoxylin and eosin (HE). For immunofluorescence analysis, sections were stained with a rabbit anti-rat CD31 antibody (1:250, Abcam, USA) and visualized using an Alexa Fluor 647 mouse-anti-rabbit IgG secondary antibody (1:500, eBioscience). For immunohistochemistry analysis, sections were stained with a rabbit anti-rat CD31 antibody (1:150, Abcam, USA) and visualized using a mouse-anti-rabbit secondary antibody (1:500, Sigma). Negative controls using IgG controls matching in species and concentration were run in parallel. Pictures were taken with a microscope (Leica CM3050S, Leica Microsystems, Germany) and a digital camera (DFC 320, Leica Microsystems) at ×100 magnification. Morphometric analysis was performed per sample followed by computer-assisted morphometric analysis (ImageJ, NIH, USA). Subsequent morphometric analyses were performed in a double-blinded manner. Four animals per group and 3 samples per animal were analyzed.

Mouse lungs were removed 12 h after LPS challenge, washed in PBS, and fixed in formalin solution 10% (v/v). The tissues were dehydrated in ethanol solutions, followed by xylene, then included in paraffin and sliced into 4 μm sections (LEICA-Instruments® CM3050, Nussloch, Baden-Württemberg, Germany). Further, the slices were stained with hematoxylin-eosin and analyzed under light microscopy (200x). The lung damage was graded taking into account the degree of parenchymal distortion in the alveolar tissue. The scores used were as follows: 0—normal, 1—in-creased thickness in <50% of interalveolar septa (IAS) due to edema and/or neutrophil infiltration, 2—increased thickness in >50% of IAS, 3—increased thickness in >50% of IAS and the presence of neutrophils within the alveolar space, and 4—consolidated infiltration of neutrophils with distortion of normal alveolar architecture. The mean score was reported for each microscope section.

After FMW, hearts were dissected with a surgical knife at room temperature, embedded into a super cryo-embedding medium (SCEM, Section Lab Co. Ltd, Hiroshima, Japan), frozen in liquid N₂, and stored at −80 °C. We prepared five sets of short-axis (transverse) tissue sections where each set consisted of a thick 450 μm “block” for quantification of metabolites and an adjacent 8 μm thin “section” for MALDI imaging analyses (see Supplementary Fig. S6). The apical two-thirds of the left ventricle in sham-operated hearts was subdivided into four short-axis blocks.
The thin sections were cut with a cryomicrotome (CM3050, Leica Microsystems) and thaw-mounted on an indium thin oxide-coated glass slide (BrukerDaltonics, Germany) at −16 °C. Heart tissues subjected to FMW tended to be more fragile than those treated by other methods, often making tissue sectioning challenging. However, embedding the tissue with SCEM medium, which did not interfere with the ionization efficiency of metabolites, helped achieve successful sectioning.

Blood was collected from the heart under anesthesia and followed by the transcardial perfusion of heparinized phosphate-buffered saline. The brain was removed and halved according to the cerebral longitudinal fissure. One half of the brain was soaked in 4% of paraformaldehyde for 2 days and then in 15 and 30% sucrose solutions for 1 day each. Brains were embedded in OCT compound, and 30 µm thick frozen brain sections (sagittal plane) were prepared using a cryostat (CM3050; Leica Microsystems, Heidelberger, Germany). The liver and gastrocnemius muscle were also collected. Blood samples were left to stand at room temperature for 30 min and then centrifuged at 1200× g at 4 °C for 20 min.

MALDI imaging analyses were performed as described previously (54 (link)). Briefly, thin sections (8 μm) of tumor were prepared with a cryomicrotome (Leica Microsystems; CM3050). Sections were attached onto indium tin oxide-coated glass slides (Bruker Daltonics GmbH) and coated with 9-aminoacridine as the matrix (10 mg/mL, dissolved in 80% ethanol) by manual spraying with an artistic brush (Procon Boy FWA Platinum, Mr. Hobby). The matrix was applied simultaneously to multiple sections to maintain consistent analyte extraction and cocrystallization conditions. MALDI imaging was performed using an Ultraflextreme MALDI-TOF/TOF mass spectrometer equipped with an Nd:YAG laser. Data were acquired in the negative reflection mode, with raster scanning at a pitch distance of 50 μm. Each spectrum was the result of 300 laser shots per data point. For TOF/TOF measurement, signals between m/z 50 and 1000 were collected. Image reconstruction for both procedures was performed using FlexImaging 4.1 software (Bruker Daltonics). Molecular identification was based on an accurate m/z value derived from FT-ICR-MS data and previous studies (54 (link)).