Fleximaging v4

Sequential adrenal sections for MALDI-MSI experiments were used for immunostaining. CYP11B2 immunohistochemistry was performed under standardized conditions on a Discovery XT automated stainer (Ventana Discovery XT Systems, Ventana Medical Systems, Inc, Tucson) using monoclonal antibodies against human CYP11B2 (diluted 1:100, a gift from Prof Celso Gomez-Sanchez, University of Mississippi), and detected by the Discovery DAB Map Kit (Roche Diagnostics/Ventana Medical Systems), including incubation with anti-mouse and anti-rabbit ready-to-use universal secondary antibodies (catalog 760-4205; Roche Diagnostics/Ventana Medical Systems). APCC regions were annotated according to the CYP11B2 immunostaining. The average spectral data of annotated APCC regions were exported from FlexImaging v. 4.0 (Bruker Daltonics) and used for further bioinformatics analysis.

FFPE adrenal samples were cut into 3-μm sections on a microtome (HM 355S, Microm; ThermoScientific) and mounted onto indiumtin-oxide-coated glass slides. The FFPE sections were incubated at 60°C for 1 hour, deparaffinized in xylene (2×8 minutes), and dried on a hot plate at 37°C. The matrix solution consisted of 10 mg/ mL 9-aminoacridine hydrochloride monohydrate (Sigma-Aldrich, Germany) in water/methanol 30:70 (v/v). SunCollect automatic sprayer (Sunchrom, Friedrichsdorf, Germany) was used for matrix application. The flow rates were 10, 20, 30, and 40 μL/min, respectively, for the first 4 layers. The other 4 layers were performed at 40 μL/min. The MALDI-MSI measurement was performed on a Bruker Solarix 7T FT-ICR-MS (Bruker Daltonik, Bremen, Germany) in negative ion mode using 50 laser shots per spot at a frequency of 1000 Hz. The MALDI-MSI data were acquired over a mass range of m/z 50 to 1000 with 50-μm lateral resolution. After MALDI-MSI measurements, acquired data underwent spectra processing in FlexImaging v. 4.0 (Bruker Daltonics, Bremen, Germany) and SCiLS Lab v. 2019 (Bruker Daltonics). MALDI-MSI data were normalized to the root mean square of all data points.

MALDI-FTICR-IMS analyses were conducted using a 7T solariX XR system (Bruker Daltonics). To detect lipid species, the continuous accumulation of selected ions (CASI) function was used in positive ion mode over a mass range m/z 500-900 with a resolution of 590,000 at m/z 400. A Smart Beam II laser was operated at 2,000 Hz, a laser power of 50%, a laser diameter of 50 µm, and a raster width of 80 µm. The SM peak obtained was accumulated and split by collision-induced dissociation (CID)-fragmentation with CASI function to obtain structural information. Mass spectra were analyzed and obtained using Data Analysis v4.3 (Bruker Daltonics).
MALDI-TOF-IMS analyses were undertaken using ultrafleXtreme (Bruker Daltonics). To detect lipid species, a reflector positive mode was used over a mass range m/z 0-1,000. A Smart Beam II laser was operated at 2,000 Hz, a laser power of 80%, a laser diameter of 100 µm, and a raster width of 100 µm. Mass spectra were analyzed and obtained using FlexAnalysis v3.3 (Bruker Daltonics).
After acquisition of mass spectra, FlexImaging v4.0 (Bruker Daltonics) was used for data processing and image generation. All imaging data were normalized by the root means square method. Lipid identification was undertaken using the MS analysis tool in the LIPID MAPS website (www.lipidmaps.org/tools/index.html).