Effects of Different Fixatives over Different Fixation Times, Including Antigenfix, on Immunohistochemical Studies

Tissue fixation is an essential step in the performance of ancillary studies, including immunohistochemistry. The aim of this study was to compare the effect of various fixatives and fixative times on immunohistochemistry (IHC) in bovine and porcine endometrium using progesterone receptors and SOX2 antibodies. Immunohistochemical staining with progesterone receptors and SOX2 was performed on tissue samples fixed in formalin, Antigenfix, Greenfix, Bouin’s solution and methacarn at 1.5, 8.5, 15.5, 29.5, 64.5, 189.5, 249.5, 309.5 and 369.5 days of fixation. Formalin and Antigenfix proved to be the best fixative for both short-term and long-term fixation for IHC. Bouin’s solution was partially applicable for short-term (24 h) fixation. Greenfix and methacarn were absolutely inappropriate fixatives for IHC (completely negative staining using methacarn). These results were obtained on an automated immunostainer using EDTA buffer pH 8.4 as the antigen retrieval solution. When the staining procedure was performed manually and citrate buffer with different pH values was used, weakly positive results were obtained with both progesterone receptors (pH 8 and 9) and SOX2 (pH 7.2); however, significantly lower staining quality was obtained using the methacarn fixative compared to aldehyde fixatives. The reported findings demonstrated the superiority of aldehyde fixatives (formalin and Antigenfix) over alcohol fixatives (methacarn) and fixatives combining both denaturing and cross-linking proteins (Greenfix and Bouin’s solution) for IHC. Antigen retrieval-IHC using EDTA buffer was found to be excellent for aldehyde fixatives, but proved to be completely unsuitable for methacarn, Greenfix, and Bouin’s solution.