Acta Vet. Brno 2022, 91: 179-188

Effects of different fixatives over different fixation times, including Antigenfix, on immunohistochemical studies

Jiří Lenz1,2,3, Dominika Macháčová2, Petra Konečná2, Luděk Fiala1,4,5, Michal Kyllar6, František Tichý2

1Cytohisto s.r.o., Břeclav, Czech Republic
2University of Veterinary Sciences Brno, Faculty of Veterinary Medicine, Department of Anatomy, Histology and Embryology, Brno, Czech Republic
3Znojmo Hospital, Department of Pathology, Znojmo, Czech Republic
4Charles University Pilsen, Faculty of Medicine, Psychiatric Clinic, Department of Sexology, Pilsen, Czech Republic
5Charles University Prague, First Faculty of Medicine, Institute of Sexology, Prague, Czech Republic
6University of Veterinary Medicine Vienna, Institute of Morphology, Department of Pathobiology, Vienna, Austria

Received August 25, 2021
Accepted February 1, 2022

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.


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