Cryostat microtome

Stems of 6‐month soil‐grown OE, WT and SE BplMYB46 birch plants were fixed in FAA solution (70% ethanol: glacial acetic acid: formaldehyde; 90: 5: 5, v/v) and embedded in frozen sectioning medium (OCT; Thermo Scientific, Waltham, MA) to obtain 25‐μm‐thick stem base sections using a Microtome Cryostat (Thermo Scientific HM560). The stem sections were stained with phloroglucinol‐HCl and toluidine blue and examined by light microscopy (Zhong et al., 2006). Lignin autofluorescence was observed under a confocal laser microscope (Zeiss, Jena, Germany). For scanning electron microscopy (SEM), 0.2‐cm‐thick sections of 12‐month soil‐grown OE, WT and SE BplMYB46 birch plants were obtained manually and observed under a scanning electron microscope (S‐4800, HITACHI, Tokyo, Japan). The ratio of vessel area to total area was measured from 12 anatomical sections representing each genotype after staining with phloroglucinol‐HCl. The secondary wall thickness of xylem fibres in the SEM micrographs was quantified in 45 cells using Image J software (http://rsbweb.nih.gov/ij/).

As described previously (23 (link)), adrenal glands were sliced into 6-µm thick sections using the cryostat microtome (Thermo Scientific) and sections were place in ice-cold phosphate-buffered saline (PBS). Tissue sections were permeabilized with PBS including 0.1% Tween detergent for 5 minutes. Tissues were blocked with 5% goat serum and then incubated with the 1:100 diluted CYP11B1 (RRID: AB_3076259) and CYP11B2 (RRID: AB_3076258) antibodies (Gomez-Sanchez) in 1% bovine serum albumin (w/v) in PBS-T overnight at 4 °C. Sections were washed with PBS 3 times and incubated with 1:500 diluted antimouse (RRID: AB_2338902)and antirabbit (RRID: AB_2338078) Alexa 647 (Jackson), respectively. For nuclear counterstaining, 20 µM Hoechst 33342 (Invitrogen H3570) was used. After washing 3 times, coverslips were mounted onto the slides with mounting medium FluorSave (Millipore 345 789).

Immunohistochemical (IHC) staining was performed as described [18 (link)]. Briefly, tumor tissues and lungs were removed and fixed with 4% paraformaldehyde and then transferred to 30% sucrose. Tissues were sectioned at 10 μm thickness using a Cryostat Microtome (Thermo Scientific). Tumor tissues were stained with phosphorylated p38γ using Diaminobenzidine (DAB) kit. Briefly, tissues were incubated with 0.3% H₂O₂ in methanol for 30 min at room temperature and then treated with 0.1% TritonX-100 for 10 min. The sections were washed twice with PBS and then blocked with 1% BSA and 0.05% TritonX-100 for 1 hour. The sections were incubated with phospho-p38γ antibody (1:300) overnight at 4°C. Negative controls were performed by omitting the primary antibody. After being rinsed in PBS, sections were incubated with a biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for an hour at room temperature. The sections were washed three times with PBS and incubated with in an avidin-biotin-peroxidase complex (1:100) for an hour and developed in 0.05% 3,3′- Diaminobenzidine (Invitrogen) containing 0.003% H₂O₂ in PBS. To examine the lung metastasis, the sections of lung tissues were stained with eosin and images were recorded using an Olympus BX51 microscope.

MLNs were removed and embedded in 2.5% medium viscosity, carboxymethyl cellulose (Sigma-Aldrich) in distilled water prior to snap freezing in a slurry of ethanol and crushed dry ice, then stored at −80 °C until processing for MSI and histological analysis. Six micrometre (μm) thick sections were cut from frozen tissue using a cryostat microtome (Thermo Scientific). Sections were cut in a specific order for MSI and histology techniques: three consecutive sections were cut for haematoxylin and eosin staining or immunohistochemistry (IHC) and mounted onto glass slides; then two adjacent sections were cut for MSI and thaw mounted onto conductive indium tin oxide (ITO) coated slides (Bruker Daltonics) for MALDI-MSI and MALDI FTICR MS, and normal non-conductive microscope slides for DESI-orbitrap-MSI. This cutting sequence was repeated until a sufficient number of slides for MSI were obtained. All slides were stored at −80 °C following sectioning.

For histology analysis, we imbedded the fixed livers in paraffin, then cut thin sections, and following standard procedures, stained them with hematoxylin and eosin. We quantified the liver histological score of inflammation according to Ishak inflammation score [43 (link), 44 (link)]. For immunofluorescence analysis, we cut frozen sections into 8μM slices in a cryostat microtome (Thermo) at -20°C and permeabilized and blocked them. We probed the tissues with appropriate, fluorescently labelled antibodies, including APC-conjugated anti-CD11b, PE-conjugated anti-CD68, and PE-conjugated anti-Ly6G (Biolegend). Finally, we dried the sections and observed them with a Nikon A1 confocal microscope.