Advisory Committee Chair
William E Grizzle
Advisory Committee Members
Stephen A Watts
Upender Mann
Document Type
Thesis
Date of Award
2007
Degree Name by School
Master of Science (MS) College of Arts and Sciences
Abstract
Because of many unanswered questions in this area, we investigated the effects of formalin fixation and the steps in tissue processing on immunohistochemical detection of specific antigen-antibody combinations of two cell cycle markers; proliferating cell nuclear antigen (PCNA) and Ki67/MIB-1. As a model to study these effects, we used cultured cells fixed in 10% neutral buffered formalin (NBF) alone and fixed in 10% NBF plus separately, the alcohols, xylene and paraffin used in each individual step in tissue processing. We found that in staining for PCNA, a proliferation antigen requires some fixation but that fixation with 10% NBF does not strongly affect the immunohistochemical signal for PCNA, except for fixation times longer than 48h, after which the signal decreases. The steps in tissue processing do decrease the intensity of PCNA staining, specifically the hydrophobic environment created by xylene. After antigen retrieval, staining for PCNA is not affected by the length of fixation but is ii decreased somewhat by dehydration at absolute ethanol and xylene during tissue processing. The Ki67/MIB-1 antibody recognizes and binds to the Ki67 epitope in cells. The frequency of this binding is a measure of cell proliferation in all active phases of the cell cycle (G1, S, G2 and M phase). While there is some decrease in the immunohistochemical signal with fixation in 10% NBF, dehydration and the development of a hydrophobic environment during tissue processing causes most of the decrease in Ki67/MIB-1 signal. After antigen retrieval, staining for Ki67/MIB-1 is decreased following periods of fixation in 10% NBF for longer than 24h. Staining for Ki67 after antigen retrieval is also decreased by dehydration during tissue processing especially the hydrophobic environment xylene. These results indicate that both fixation and the steps of tissue processing - dehydration through absolute ethanol and hydrophobic environment in xylene - affect epitope recognition by specific antibodies.
Recommended Citation
Otali, Dennis, "The Combined Effect Of Formalin Fixation And Individual Steps In Tissue Processing On Immunorecognition" (2007). All ETDs from UAB. 3618.
https://digitalcommons.library.uab.edu/etd-collection/3618