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Updated: May 14, 2026
Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
Published on: November 14, 2016
Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol
1Departments of Pathology.
Insights
Prolonged formalin fixation degrades antigen detection. Transferring cells to ethanol after short formalin fixation preserves immunorecognition for key cancer biomarkers.
Area of Science:
- Histopathology and Immunohistochemistry
- Cell Biology
- Cancer Research
Background:
- Formalin fixation is standard for tissue preservation but can impair antigen-antibody binding (immunorecognition).
- Short formalin fixation followed by ethanol storage is a proposed method to mitigate fixation-induced antigen masking.
- The impact of this fixation-ethanol transfer method on immunorecognition requires further investigation.
Purpose of the Study:
- To evaluate the effect of prolonged 10% neutral buffered formalin (NBF) fixation on immunorecognition of specific biomarkers.
- To assess the efficacy of transferring fixed cells to 70% ethanol in preserving immunorecognition.
- To compare the impact of different fixation and storage durations on antigen detection in cancer cell lines.
Main Methods:
- DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were fixed in 10% NBF for varying durations (5 min to 180 h).
- Separate cell aliquots were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3 to 168 h.
- Immunostaining was performed for Proliferating Cell Nuclear Antigen (PCNA), Ki67-MIB-1, cytokeratins AE1/AE3, and Epidermal Growth Factor Receptor (EGFr).
Main Results:
- Immunorecognition for PCNA, Ki67-MIB-1, and cytokeratins AE1/AE3 significantly decreased between 18 and 36 h of NBF fixation.
- Complete loss of PCNA immunorecognition and substantial loss of Ki67-MIB-1 and cytokeratin detection occurred after 108-180 h of NBF fixation.
- EGFr showed less sensitivity to prolonged NBF fixation.
- Transfer to 70% ethanol after 12 h of NBF fixation successfully preserved immunorecognition for all tested antibodies.
Conclusions:
- Prolonged formalin fixation (>18 h) significantly compromises the detection of key cellular markers like PCNA, Ki67, and cytokeratins.
- The 10% NBF fixation followed by storage in 70% ethanol is an effective strategy to maintain antigen integrity and immunorecognition.
- This method offers a viable alternative for preserving tissue samples for immunohistochemical analysis, especially for sensitive antigens.
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.
