OBJECTIVE: To evaluate the influence of pH values of fixatives and antigen retrieval buffers on immunostains. METHODS: Samples of normal skin, gastric mucosa, uteri leiomyoma and breast carcinoma were fixed in buffered formalin with different pH values, namely 3.0, 5.0, 7.0, 8.0 and 10.0 to detect EMA, SMA, ER and CK (AE1/AE3) expression. Evinision two-step method was employed. Antigen retrieval was achieved by microwave using sodium citrate solution with pH 6.0, 7.0, 8.0 or pH 8.0 EDTA solution respectively. RESULTS: The number and intensity of positive cells varied due to different conditions. The change of positive cell's number is important for CK (AE1/AE3) expression, and the change of positive intensity is important for SMA and EMA expression. CONCLUSIONS: (1) The best results could be obtained by the use of 10% neutral or alkalescent buffered formalin. (2) Antigen retrieval effect is better in the solution of pH 7.0, 8.0 than in that of pH 6.0. EDTA(pH 8.0) appears better for skin. The best effect of Estrogen Receptor needs pH 8.0 sodium citrate solution. (3) The background could be easily induced by the fixative over pH 8.0. Antigen retrieval solution over pH 8.0 could result in detachment.
OBJECTIVE: To evaluate the influence of pH values of fixatives and antigen retrieval buffers on immunostains. METHODS: Samples of normal skin, gastric mucosa, uteri leiomyoma and breast carcinoma were fixed in buffered formalin with different pH values, namely 3.0, 5.0, 7.0, 8.0 and 10.0 to detect EMA, SMA, ER and CK (AE1/AE3) expression. Evinision two-step method was employed. Antigen retrieval was achieved by microwave using sodium citrate solution with pH 6.0, 7.0, 8.0 or pH 8.0 EDTA solution respectively. RESULTS: The number and intensity of positive cells varied due to different conditions. The change of positive cell's number is important for CK (AE1/AE3) expression, and the change of positive intensity is important for SMA and EMA expression. CONCLUSIONS: (1) The best results could be obtained by the use of 10% neutral or alkalescent buffered formalin. (2) Antigen retrieval effect is better in the solution of pH 7.0, 8.0 than in that of pH 6.0. EDTA(pH 8.0) appears better for skin. The best effect of Estrogen Receptor needs pH 8.0 sodium citrate solution. (3) The background could be easily induced by the fixative over pH 8.0. Antigen retrieval solution over pH 8.0 could result in detachment.