Understanding gastric structure is essential for studying gastrointestinal disorders. While traditional histological analysis focuses on two dimensions, micro-computed tomography ( \(\mu\) CT) offers high-resolution three-dimensional (3D) imaging but requires staining for proper visualization of soft tissues. This study presents the first detailed 3D anatomical reconstruction of the rat stomach using \(\mu\) CT, investigating optimal preparation and staining protocols to enhance image quality and ultimately help understand structural-functional relationships in rat stomachs. Fifteen rat stomachs were dissected and prepared through a five-stage process: fixation (2% neutral buffered formalin—NBF), dehydration (graded ethanol), staining (0.75% alcoholic Lugol’s solution and 2% alcoholic phosphotungstic acid—PTA), and \(\mu\) CT imaging (SkyScan 1172, Bruker, Belgium). Images were reconstructed using InstaRecon and NRecon software (Bruker, Belgium). Quantitative comparison using signal-to-noise (SNR) and contrast-to-noise (CNR) ratios indicated improved performance with PTA under stain-optimised imaging parameters (CNR: \(3.8\pm 0.4\) vs. \(2.9\pm 0.3\) ; SNR: \(13.7\pm 1.3\) vs. \(10.1\pm 1.3\) ; p < 40.05), but also revealed tissue shrinkage (21.5% area reduction, 33.1% volume reduction). Fixation duration (3–17 weeks) minimally impacted image quality. Optimal staining duration of 7–9 weeks maximized contrast, while prolonged staining (> 14 weeks) caused tissue tearing. In conclusion, both iodine and PTA stains highlighted useful anatomical features for analysis. Specifically, we recommend 2% NBF fixation for three weeks, followed by 7–9 weeks of PTA staining for high-quality \(\mu\) CT imaging of rat gastric tissue.
