Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Abstract

Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.

Key words::

• cytokeratins AE1/AE3
• EGFr
• 70% ethanol
• 10% formalin
• immunohistochemistry
• Ki67-MIB-1
• PCNA

Acknowledgments

Supported in part by the following grants: the Susan G. Komen Breast Cancer Foundation (BCTR0600484), the Cooperative Human Tissue Network (5U01CA44968), the UAB Pancreatic (2P50CA101955) and Breast (5P50CA089019) SPORES, the DOD Grant (W81XWH-10-1-0543), the UAB Comprehensive Cancer Center Core Support Grant (P30CA13148) and the U54 MSM/TU/UAB Comprehensive Cancer Center Partnership (2U54CA118948, 2U54CA118623).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.