GSM9336189: adrenal gland -male; Mus musculus; OTHER
FFPE tissue blocks were sectioned at 5 µm thickness using a Leica RM2255 microtome. Four male adrenal sections were placed on a single Visium HD capture area (6.5mm × 6.5mm),  and four female adrenal sections were placed on a separate Visium HD capture area. This spatial pooling design enabled simultaneous profiling of multiple biological replicates while preserving spatial information for each individual tissue section. Sections were mounted on Visium HD slides and stored at 4°C. RNA quality was assessed using adjacent sections with Agilent 2100 Bioanalyzer; only samples with DV200 > 50% were processed. Slides were deparaffinized in xylene, rehydrated through graded ethanol series, and stained with H&E. High-resolution brightfield images were acquired at 20× magnification using Grundium Ocus 40 scanner (~300 DPI) for cell segmentation and spatial alignment. Spatial transcriptomic profiling was performed using 10x Genomics Visium HD Spatial Gene Expression workflow for FFPE samples with CytAssist instrument (User Guide CG000684, Rev B). Each capture area (whole male, whole female) was processed independently. Protocol included: enzymatic permeabilization, RNase H-mediated probe cleavage, reverse transcription with spatially barcoded primers (bin size: 8 µm), second-strand synthesis, and cDNA amplification. Libraries were prepared according to Visium HD Spatial Gene Expression Library Preparation protocol. Each library was quantified using Qubit fluorometer, normalized, and diluted to 650 pM final concentration.