Objective To investigate the effect of antigen retrieval time and pH value of the retrieval solution on the immunohistochemical staining results of Pax-8 and CD10. Methods Fifteen surgical specimens of kidneys and livers （15 cases respectively） were selected,and the EnVision two-step immunohistochemical method was used to perform antigen retrieval with pH8.0 and pH9.0 in renal tissue Pax-8 and liver tissue CD10, respectively. The retrieval time was 1.5 min,2 min,2.5 min,and 3 min. The staining was evaluated by the main pathologist in a blind review. Results The positive expression of Pax-8 and CD10 in both groups of retrieval solutions was good（P< 0.05）. The double-factor analysis of variance of renal tissue Pax-8 antibody staining showed that the main effect of retrieval solution pH was significant（P< 0.01, partial η²= 0.424）. Backgroundstaining in pH 8.0 retrieval solution groups of 1.5 min, 2 min, 2.5 min, and 3 min was significantly lighter than that in pH 9.0 groups,with statistically significant difference （P< 0.01）. There was no statistically significant difference in the interaction effect of the retrieval time and the interaction effect between the two factors. For hepatic tissue CD10 antibody staining,double-factor ANOVA indicated a significant main effect of pH （P< 0.01, partial η²= 0.076）, non-significant main effect of retrieval r time （P> 0.05）, but significant interaction effect （P< 0.05, partial η²= 0.068）. The pH 8.0-1.5 min group showed significantly lighter background staining than pH 9.0-1.5 min group （P< 0.01）. Within pH 8.0 groups,no significant differences were observed across time points （P> 0.05）. Under pH 9.0 conditions, background staining progressively decreased with extended repair time, reaching the lightest level in 3-min group. Conclusions The background of Pax-8 staining n renal tissue is not related to antigen retrieval time,while pH 8.0 retrieval solution can reduce the background staining. For hepatic tissue CD10 staining,antigen retrieval duration should be appropriately adjusted according to pH variation of retrieval solutions：1.5 min for pH 8.0 and 3 min for pH 9.0.
Key words
immunohistochemistry /
antigen retrieval /
pH /
background coloration
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