Cryomicrotome

The frozen skin biopsies were cut using a cryomicrotome (Thermo Scientific, Illkirch-Graffenstaden, France) after being immersed in the cryomatrix at −50 °C. The chamber of the microtome was fixed at −20 °C and sections of 7 µm were prepared and used for the immunohistochemcial staining. The sections were fixed with 100% methanol for 5 min at −20 °C. Sections were then washed two times with phosphate-buffered saline containing 0.1% Tween 20 before being incubated with a 1% BSA solution in PBS. Samples were then washed and incubated with the primary antibody overnight at 4 °C and washed with the 0.1% Tween 20 in PBS solution. If needed, a secondary antibody was then added and incubated with the sections for 1 h at room temperature. Two steps of washing were then performed to remove the unreacted antibody; antibody dilutions were produced according to the manufacturer’s instructions. For the accurate identification of the epidermis layer, the nuclei of the cells were stained with DAPI for 10 min at room temperature before being washed and prepared for microscopical examination. The samples were examined using a confocal laser scanning microscope (CLSM) LSM 800 Zeiss (Zeiss, Marly Le Roi, France). For the determination of epidermal thickness, images were again analyzed using ImageJ software (National Institutes of Health, Bethesda, MA, USA).

After 7‐day co‐treatment of LPS and natural compounds, the mice were perfused with saline and 4% paraformaldehyde for IF. A total of 30 μm‐thick coronal brain slices (AP −2.0 and −2.5 mm from bregma) were obtained using cryomicrotome (Thermo Fisher). IF was conducted as previously reported,
⁶ (link) with the following primary antibodies: anti‐GFAP (chicken polyclonal, 1:1000; MilliporeSigma, AB5541), anti‐GABA (guinea pig polyclonal, 1:500; MilliporeSigma, AB175) and anti‐MAO‐B (rabbit polyclonal, 1:1000; Abcam, AB137778). Confocal images (60x) were obtained with a multiphoton laser scanning microscope (Nikon A1R, Tokyo, Japan) and analyzed by Imaris 9.3 software.

Leaves, stems, roots and cluster roots of living plants of G. meisneri were carefully washed with deionized water before being dried with absorbing paper. Samples were cut and fragments of about 1 cm long were immediately immersed in an embedding compound (Optimal Cutting Temperature OCT from VWR) and fast frozen in isopentane, as liquid cryogen, cooled by liquid nitrogen^{90 (link)}. This cryofixation protocol ensured an extremely fast cooling of the samples, preventing ice crystal formation. Therefore, it limited damage to the cellular structures and preserved elemental distribution close to its natural state. Leaf samples were divided into mid-rib (central vein) and margin. For the roots, the primary root and cluster roots were sampled. The samples were transported from New Caledonia in a cryogenic container, kept at − 80 °C using dry ice, to SOLEIL Synchrotron (Saint-Aubin) for analysis and kept in a freezer at − 80 °C before cryo-sectioning.
Transverse sections of frozen-hydrated tissues were cut at a thickness of 20 μm using a cryo-microtome (Thermo Fisher Scientific). The cryo-chamber was kept at − 20 °C. The thin sections were placed between two Ultralene films (SPEX SamplePrep) in a copper sample holder.

Mice treated with SL. pNASGFP or PBS (control) were sacrificed on the indicated days after bacterial injection. Tumor tissues were embedded in optimal cutting temperature (OCT) compound (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C. Frozen tumor sections (5 μm thick) were made with a cryomicrotome (Thermo Fisher Scientific, Waltham, MA, USA) and mounted onto glass slides. The slides were blocked with 5 % BSA at room temperature for 1 h, and then incubated overnight at 4 °C with mouse anti-iNOS monoclonal antibody (1:100, Invitrogen, Waltham, MA, USA). After three washes with PBS containing 0.5% Tween-20 (PBS-T), the slides were incubated with Alexa 555-conjugated anti-Rat Ig antibody (1:200, Thermo Fisher Scientific, Waltham, MA, USA) in the dark for 1 h. After washing with PBS-T, the slides were mounted in ProLong antifade mounting solution with 4′,6-diamidino-2-phenylindole dye (DAPI, Invitrogen, Waltham, MA, USA) and sealed with nail polish. The fluorescence signals of sfGFP (green), iNOS (red), and DAPI (blue) were detected using an LSM510 fluorescence microscope (Zeiss, Jena, Germany), and processed using LSM image software.

Normal and injured spinal cord tissues were collected at different time points after injury. The spinal cord was excised, frozen, and cut into slices (15 µm thick) using a cryomicrotome (Thermo Fisher Scientific, USA). The specimens were mounted on Ultralene foil and freeze-dried. The elements in the injured areas of the mouse spinal cord were detected using the synchrotron radiation-based micro-X-ray fluorescence (SR-µXRF) technique at BL15U1, Shanghai Synchrotron Radiation Facility (SSRF), China. The specimens were fixed to the platform at a 45° angle to the incident SR X-ray beam, and a silicon drift detector (SSD) (Vortex, USA) was placed at a 90° angle to the incident SR X-ray beam for recording the fluorescent counts of the metals. The energy of the exciting beam was set to 10 keV. The specimens were fixed on a sample platform controlled by a motorized X-Y mapping stage and were continually scanned in steps of 4 µm. At each point of the line scan, a fluorescent spectrum was recorded for 12 s with a resolution of 4 µm × 2 µm. The PyMCA program analyzed the SR X-ray spectra [18 (link)]. The intensity maps for the elements in the injured area of the spinal cord were analyzed using the Igor Pro software (WaveMetrics, USA).

From each rat, the same part of the liver tissue was fixed in 4% paraformaldehyde for over 48 h. The liver sample was dehydrated in gradient alcohol, embedded in paraffin wax, and then sliced into 4-μm thick sections and stained with hematoxylin-eosin (H and E) for observation. Each section was observed with a microscope at ×200 magnification (Leica DM4000B, Solms, Germany).
The frozen liver tissue was sliced into 8-μm thick sections using a cryomicrotome (Thermo Fisher Scientific Inc, Massachusetts, United States), fixed in fixing solution (Servicebio Technology Co., Ltd, Wuhan, China) for 15 min, and stained with oil red O solution (Servicebio Technology Co., Ltd, Wuhan, China) for 15 min, The liver tissues were washed with 60% isopropanol, and then the nuclei were then stained with hematoxylin staining solution (Servicebio Technology Co., Ltd, Wuhan, China). The slices were observed, and photos were taken using an optical microscope (Olympus, Tokyo, Japan).