To evaluate the potential effect of embedding with carboxylmethyl cellulose (CMC) and embedding with optimal cutting temperature (OCT) compound followed by washing with PBS (OCT-W) on the analysis of breast cancer tissue samples with desorption electrospray ionization mass spectrometry imaging (DESI-MSI).DESI-MSI of fresh frozen (FF) tissue samples, OCT-embedded samples, CMC-embedded samples, and OCT-W samples from the same breast cancer tumor tissue were performed. The ratio of maximum abundance ion was used to assess the reproducibility of DESI-MSI analysis. In addition, the effects of the treatment of each group were examined by comparing the characteristic ion species and the ion signal intensity detected by DESI-MSI.DESI-MSI of continuous sections of FF samples showed that the coefficient of variation (CV) of the pair-to-pair ratios of m/ z 281.25, m/ z 309.28 and m/ z 279.23 ions, the three ions with the highest intensity in the tumor region, were 19.61%, 20.74% and 10.18%, respectively. The characteristic ion species detected by DESI-MSI of CMC embedded tissue and the OCT-W tissue were almost the same, compared with those of the FF tumor tissue. However, ion species detected in OCT embedded samples were less than 50% of the FF samples. In terms of ion signal intensity, the CMC embedded tissue was not affected overall, while the signal of most of the characteristic ions of the OCT-W group showed decreased intensity (P<0.05).FF tissue sections and CMC-embedded samples can be used for DESI-MSI routine analysis. OCT embedding affects the feasibility of sample analysis whether or not the sample undergoes washing with PBS. CMC embedding agent is recommended if the tissue sections need to be fixated and supported due to small sample size, fragility, or other problems.Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).