We investigated the impact of immunohistochemistry (IHC) pre-treatment steps on antigens.Salmonella typhimurium was selected as the observed antigen. The antigen was subjected to IHC pre-treatment steps involving a series of reagents, including 10% formaldehyde, ethanol, and xylene. Antigenicity was then measured by agglutination reaction.The agglutination titer for S. typhimurium was higher in the untreated control group than in the experimental group, indicating that pre-treatment inhibited antigen activity. The inhibitory effect of ethanol was greater than that of 10% formaldehyde and xylene. Unexpectedly, partial antigen recovery can be achieved from a preparation of paraffin section after hydration.S. Antigens may be strongly inhibited (inhibition: 70.8%) by IHC pre-treatment steps, especially by alcohol treatment. There is an experimental foundation for antigen retrieval in IHC.