Immunohistochemical Analysis of SHMT2

The expression and location of SHMT2 were estimated with IHC by the streptavidin peroxidase complex method according to the method described in a previous study [12 (link)]. In brief, after being deparaffinized and rehydrated with xylene and graded alcohol, tissues were incubated in boiled 0.01 M citrate buffer (pH=6.0) for the best antigen retrieval. We used 3% H₂O₂ to inactivate the endogenous peroxidase. Following the blockage of unspecific binding by 5% bovine serum albumin (BSA), tissues were incubated in primary antibody of SHMT2 at 1: 100 (Abcam, Cambridge, MA, USA, cat. no. EPR3198) at 1: 100 dilution at 4°C overnight. After rinsing in phosphate-buffered saline 3 times, tissues were incubated in HRP-labeled secondary antibody (ZSBio, Beijing, China) at room temperature for 30 min. Finally, the complex reagent of streptavidin peroxidase (ZSBio, Beijing, China) was used, and 3,3′-diaminobenzidine solution (ZSBio, Beijing, China) was applied for final visualization of the antigen.