THE COMBINED EFFECT OF FORMALIN FIXATION AND INDIVIDUAL STEPS IN TISSUE PROCESSING ON IMMUNORECOGNITION DENNIS OTALI

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1 THE COMBINED EFFECT OF FORMALIN FIXATION AND INDIVIDUAL STEPS IN TISSUE PROCESSING ON IMMUNORECOGNITION by DENNIS OTALI WILLIAM E. GRIZZLE, COMMITTEE CHAIR STEPHEN A. WATTS UPENDER MANNE A THESIS Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Master of Science BIRMINGHAM, ALABAMA 27

2 THE COMBINED EFFECT OF FORMALIN FIXATION AND INDIVIDUAL STEPS IN TISSUE PROCESSING ON IMMUNO-RECOGNITION DENNIS OTALI Biology (MS) 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 1% neutral buffered formalin () alone and fixed in 1% 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 1% 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

3 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 1%, 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 1% 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. iii

4 ACKNOWLEDGEMENTS I would like to thank my mentor Dr William E. Grizzle for the untiring effort to see this thesis completed. At those times when I lost faith in myself, he always offered words of encouragement that saw me through. His passion and love for science has also had a special effect on me, and I hope to emulate him in my career. For Dr Upender Manne, few words can express my appreciation for his help; I will always be indebted to him. Dr Watts was always the teacher and had an answer to all my concerns. I specially want to acknowledge Cecil R. Stockard and Denise Oelschlager for initiating me into the field of immunochemistry and cell culture respectively and Dr. Wen Wan for the statistical analysis. I would also wish to extend my appreciation to my sponsors, Fogarty International, without whose financial support I wouldn t have embarked on this journey and my employers, Makerere University Kampala, Uganda for granting me a study leave. Finally, I would like to thank my family, especially my wife, for the unconditional support during these two years while I was away from home and those special friends who always stood by me. iv

5 TABLE OF CONTENTS ABSTRACT... ii ACKNOWLEDGEMENTS... iv LIST OF FIGURES... vii LIST OF ABBREVIATIONS... xi INTRODUCTION... 1 FIXATION... 4 What is Fixation?... 4 Effects of Fixation... 4 Types of Fixation... 5 Formaldehyde Fixation... 6 Alcohol Fixation... 8 TISSUE PROCESSING... 1 DETECTION SYSTEMS ANTIGEN RECOVERY METHODOLOGY Slide Preparation:... 2 Cellular Preparation: Immunostaining: Effects of Tissue Processing: Summary of Tissue Processing: Analysis RESULTS... 3 General Observations Related to the Tissue Model :... 3 Staining of DU145 Cells for Ki67: Comparison of Ki67 Staining in DU145 and SKOV3 Cells: v

6 Staining of DU145 Cells for PCNA: Comparison of PCNA Staining in DU145 and SKOV3 Cells: Comparison of Experimental Designs I and III: Slide Preparation DISCUSSION REFERENCE APPENDIX A Staining Procedure For Paraffin Sections-Biogenex Concentrated HRP Kit vi

7 LIST OF FIGURES Figure 1: shows a template used to calculate cell numbers...29 Figure 2 :shows a template used to determine percentages of cells per staining category29 Figure 3 displays figures derived from calculating the staining score...29 Figure 4 Effects of various times of fixation in 1% with or without AR on immunostaining of Ki67 in DU145 cells...4 Figure 5: Immunostaining score (Ki67 in DU145 cells) vs time of fixation in 1% 4 Figure 6: Effects of various times of fixation in 1% plus 7% ethanol (1h) with and without AR on immunostaining of Ki67 in DU145 cells...41 Figure 7: Immunostaining score (Ki67 in DU145 cells) vs time of fixation in 1% plus 7% ethanol...41 Figure 8: Effects of various times of fixation in 1% plus 7%, 8%, 9% ethanol (1h each) with and without AR on immunostaining of Ki67 in DU145 cells...42 Figure 9: Immunostaining score (Ki67 in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration Figure 1: Effects of various times of fixation in 1% plus 7%, 8%, 9% ethanol (1h each) and 3h of absolute ethanol with and without AR on immunostaining of Ki67 in DU145 cells...43 Figure 11 Immunostaining score (Ki67 in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration and absolute ethanol 3h vii

8 Figure 12: Effects of 1% fixation for various times plus 7%, 8%, 9% ethanol (1h each), 3h of absolute ethanol, and 2h of xylene on immunostaining for Ki67 in DU145 cells with or without AR Figure 13 Immunostaining score (Ki67 in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration, absolute ethanol 3h and xylene 2h...44 Figure 14: Effects of 1% fixation for various times plus 7%, 8%, 9% ethanol (1h each), 3h of absolute ethanol, 2h of xylene and 2h of paraffin on immunostaining for Ki67 in DU145 cells with or without AR Figure 15:Immunostaining score (Ki67 in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration, absolute ethanol, 3h xylene 2h and 2h of paraffin...45 Figure 16: Comparison of immunostaining score (Ki67 in DU145 and SKOV cells) vs time of fixation in 1% Figure 17: Comparison of immunostaining score (Ki67 in DU145 and SKOV3 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration, absolute ethanol 3h and xylene...46 Figure 18: Effects of various times of fixation in 1% with or without AR on immunostaining of PCNA in DU145 cells Figure 19: Immunostaining score (PCNA in DU145 cells) vs time of fixation in 1%...47 Figure 2 Effects of various times of fixation in 1% plus 7% ethanol (1h) with and without AR on immunostaining of PCNA in DU145 cells...48 viii

9 Figure 21: Immunostaining score (PCNA in DU145 cells) vs time of fixation in 1% plus 7% ethanol...48 Figure 22: Effects of various times of fixation in 1% plus 7%, 8%, 9% ethanol (1h each) with and without AR on immunostaining of PCNA in DU145 cells...49 Figure 23: Immunostaining score (PCNA in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration...49 Figure 24 Effects of various times of fixation in 1% plus 7%, 8%, 9% ethanol (1h each) and 3h of absolute ethanol with and without AR on immunostaining of PCNA in DU145 cells....5 Figure 25: Immunostaining score (Ki67 in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration and absolute ethanol 3h....5 Figure 26: Effects of 1% fixation for various times plus 7%, 8%, 9% ethanol (1h each), 3h of absolute ethanol, and 2h of xylene on immunostaining for PCNA in DU145 cells with or without AR Figure 27: Immunostaining score (PCNA in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol 1h at each concentration, absolute ethanol 3h and xylene 2h...51 Figure 28: Effects of 1% fixation for various times plus 7%, 8%, 9% ethanol (1h each), 3h of absolute ethanol, 2h of xylene and 2h of paraffin on immunostaining for PCNA in DU145 cells with or without AR Figure 29: Immunostaining score (PCNA in DU145 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration, absolute ethanol,3h xylene 2h and 2h of paraffin ix

10 Figure 3: Comparison of immunostaining score (PCNA in DU145 and SKOV3 cells) vs time of fixation in 1% Figure 31: Comparison of immunostaining score (PCNA in DU145 and SKOV3 cells) vs time of fixation in 1% plus 7%, 8% and 9% ethanol1h at each concentration, absolute ethanol 3h and xylene...53 Figure 32: shows the comparison between experimental design I and III DU145 PCNA.54 Figure 33: shows the comparison between experimental design I and III DU145 Ki x

11 LIST OF ABBREVIATIONS AR ATCC Av cat. DAB DI EDTA h H&E HRP IHC m no. PBE Antigen retrieval American Type Culture Collection Average Catalogue 3,3 diaminobenzidine De-ionized Ethylene Diamine Tetraacetic Acid Hours Hematoxylin and Eosin Horseradish Peroxidase Immunohistochemistry minutes Neutral buffered formalin Number Phosphate buffer saline with EDTA, sodium azide and bovine serum albumin PBS PCNA Phosphate buffered saline Proliferating Cell Nuclear Antigen xi

12 INTRODUCTION Immunohistochemistry (IHC) is one of the more frequently used methods to characterize the molecular features of diseases including invasive as well as preinvasive neoplastic lesions, e.g., ductal carcinoma in situ (DCIS) of the breast. Immunohistochemical methods are used as an aid in diagnosis and in determining prognosis and risk assessment as well as in evaluating therapeutically induced changes as surrogate endpoints. Thus IHC has a very important clinical impact. While immunohistochemical methods appear simple, their performance is affected by multiple interdependent variables including the preparation of tissue prior to immunostaining. Formalin, a widely used fixative for preserving specimens has been implicated in masking epitopes during IHC either via changes in conformation of a protein, blocking antibody access to epitopes and/or modification of epitopes. Such changes, which may be reversible or irreversible, frequently are hypothesized to prevent the antibody from recognizing and/or binding to an altered or hidden epitope (antigenic determinant) (Hayat, 21). Specifically, the common concept is that fixation has the major effect by causing crosslinking of the antigens thus changing the configuration of proteins or peptides and rendering epitopes undetectable or unreachable by antibodies. Actually at the currently short times of fixation (< 24h), crosslinking is probably not extensive and most changes are due to addition of reactive hydroxymethyl groups ( i.e., -CH2OH). 1

13 While the effects of fixation on epitope masking have been well documented (Islam Eltoum et al., 21a; Grizzle et al., 21; Namimatsu et al., 25; Rait et al., 24a; Rait et al., 24b), tissue processing has not been studied adequately nor has masking of epitopes been attributed to tissue processing. The steps involved in tissue processing, in addition to fixation, include dehydration in alcohol, infiltration by an organic solvent establishing a hydrophobic environment and impregnation in paraffin (Grizzle et al., 21). Because fixation and tissue processing involve multiple steps, it is clear that during one of the steps, some epitopes may be masked either by epitope modification due to the formation of large compact protein complexes, secondary to folding of the protein or binding of the reactive hydroxymethyl group of formalin close to the epitope and subsequent hiding or blocking the epitope from antibodies (Hayat, 21). Fixation and tissue processing, over which there has been little control, may have extensive effects on the quality of immuno-stains as do, primary antibodies and of secondary detection systems (Grizzle et al., 1998). Specifically, our hypothesis was that both fixation plus the steps of tissue processing strongly affect the end results of IHC. To establish how fixation and tissue processing affect IHC, we used a cell model of cultured DU145 and SKOV3 cells grown on microscope slides to mimic tissues. In the first step, the cells were in fixed in 1% for different durations of time, then immunostained for evaluation. Subsequent sets of experiments again involved cultured cells on microscope slides fixed in 1% for some lengths of time in combination with the individual steps in tissue processing; again immunoreactivity was assessed at the end of each fixation condition in combination with each step in tissue processing. 2

14 Fixation and tissue processing were followed by immunostaining with either PCNA or Ki67/MIB-1. PCNA was chosen because it can be detected without antigen retrieval (AR) while Ki67/MIB-1 on the other hand was selected because it s detection in formalin fixed paraffin embedded tissues requires antigen retrieval. For detection of the antigen-antibody reaction, we used multi-species link and label. The advantage of these secondary reagents is that they are a cocktail of antibodies raised against immunoglobulins of different species allowing one secondary reagent to be used for both monoclonal and polyclonal antibodies (Clive R. Taylor et al., 26; Ramos-Vara, 25). We opted for the heat-induced method because it has been the most tested and is widely used for its simplicity. IHC involves many steps, in addition to the variables involved in preparation of tissues, a challenge posed because any one factor within a step might affect the attainment of consistent immunohistochemical results. 3

15 FIXATION What is Fixation? Fixation is a term used to describe the preservation of cells and tissues in a reproducible life like manner. It achieves this by preserving cellular components of both soluble and structural proteins thus preventing cellular disintegration. Because of this ability to preserve cells and tissues chemical fixation is a basis for microscopic studies. For fixation to be described as good, the cells or tissues should retain their authentic intra and extra cellular molecules as they were before addition of the fixative (Hayat, 21). Effects of Fixation The aim of fixation is to prevent autolysis of cells or tissues by lysosomal enzymes and putrefaction by bacterial and fungal growth. Fixation may induce shrinkage or swelling and hardening of tissues. Hardening is caused by tissue proteins becoming dehydrated thus creating a firmness that prevents efflux of soluble cellular constituents by rendering them insoluble while it also preserves cellular organelles. Fixation therefore prepares tissues and cells for the subsequent steps in tissue processing and eventual staining. An ideal fixative should therefore penetrate the tissue rapidly, cause little shrinkage, enhance morphological details of cells besides being relatively non toxic and 4

16 not expensive. There is no claim to an ideal fixative; therefore the choice of fixatives depends on a number of considerations such as type and nature of cells or tissues to be demonstrated and the effectiveness of the fixative in demonstrating desired characteristics. 1% has many qualities of a fixative necessary for diagnostic use in pathology and it thus has become the primary fixative used in clinical practice of pathology and in the training of pathologists. Because many pathology laboratories have large repositories of formaldehyde fixed tissues, an invaluable resource in retrospective studies of disease, the effects of 1% on immunoreactivity continues to be a subject of interest. Types of Fixation Fixation can be divided into physical and chemical fixation. Physical fixation includes fixation by heat; an example is use of heat to precipitate proteins. Another form of physical fixation, freeze drying, is useful in studying small molecules which are highly soluble. Specifically, specimens not more than 2mm thick are immersed in isopentane cooled by liquid nitrogen, and kept in a vacuum chamber at -4 C. Complete dehydration occurs through sublimation without extensive loss of morphological detail or small soluble proteins. Chemical fixation involves use of organic and inorganic reagents to maintain adequate morphological preservation. Commonly used reagents in chemical fixation are formaldehyde and alcohol (usually ethanol). Formaldehyde fixes cells or tissues by addition of reactive hydroxymethyl groups and formation of methylene bridges this is additive or non-coagulant fixation, while alcohols on the other hand, fix cells or tissues 5

17 through removal of free water from proteins in tissues and can be classified as coagulant fixatives (Islam Eltoum et al., 21b). Several factors that affect chemical fixation include; concentration of fixative, temperature of fixation, buffers and ph, duration of fixation and size of the specimen as well as osmolarity and ionic composition of fixative. Formaldehyde Fixation Formaldehyde is a gas that mixes with water to a maximum concentration of 4% by weight and thus is provided as a liquid concentration of 4%. This mixture of formaldehyde and water called formalin. It is used as 1% concentration by volume (equivalent of 4% weight by volume of formaldehyde) buffered to neutral ph. Formalin has been used in pathology during the twentieth century for fixing of tissues or cells, because it maintains features of cells optimal for histopathologic examination. For molecular purposes however, 1% is inadequate because of covalent modifications observed on nucleic acids (Paska et al., 24) affect their detection and identification. The 4% concentration of formaldehyde (1% formalin), commonly used for fixation has various forms in use depending on its effects and/or nature of tissues or cells to be fixed. Formaldehyde in aqueous solution is mainly methylene glycol (~99%) and (~1%) formaldehyde. The methylene glycol rapidly penetrates tissue, whereas formaldehyde which directly fixes the proteins in tissues penetrates much slower. (Srinivasan et al., 22) state that at very low concentration cells may be able to metabolize some fixatives, e.g. formaldehyde by aldehyde dehydrogenase. As the concentration of the fixative increases the chemical kinetics will be altered and the fixative will be in excess of the proteins. At the same time, metabolic pathways will be cut off at different times, 6

18 exerting various effects on organelles. Formaldehyde as 1% formalin reacts with free proteins forming a reactive hydroxymethyl group and this structure is active in creating crosslinking by forming methylene bridges (Fraenkel-Conrat & Olcott, 1948; Rait et al., 24b). Neutral ph favors the formation of such bridges over acid ph. At neutral ph hydrogen sites in peptide molecules are available for linkage because of their uncharged state (Hayat, 21). For that reason, salts have been added to many formalin preparations to make neutral buffered formalin () which perhaps also helps maintain the elasticity of cells and tissues as well as keep ph to a constant level. A neutral ph is also used to prevent the formation of formalin pigment which is a brown to black insoluble pigment that forms in acid ph from metabolites of hemoglobin. To use formaldehyde as a fixative for nucleic acids in tissue, it is recommended that the maximal pre-fixation time be less than 2 hours and that fixation utilize 1% at 4 C at low salt concentration and with a duration of fixation of 3 to 6 hours, ethylene diamine tetra acetic acid (EDTA) (2mmol/L to 5mmol/L) should be used as an additive to avoid a low ph environment (Srinivasan et al., 22). These recommendations are consistent with (Rait et al., 26) who report a slow rate of recovery of nucleic acids at room temperature from fresh formalin fixed specimens. For some antigens, fixation is an essential step in IHC; however the formation of methylene bridges in tissues sometimes prevents antibody recognition during IHC. In another study that looked at interrelationship between crosslinking, immunoreactivity and heat treatment, (Rait et al., 24b) show via gel electrophoresis and enzyme link assays that formalin induced crosslinking does affect tissues thus reducing immunoreactivity. They report that an extremely large concentration of proteins and other molecules within tissues leads to the formation of a 7

19 dense irregular network of crosslinks that can prevent antibody penetration to the location of its antigen. Within the tissue itself, formaldehyde may interact to shield epitopes on target antigens through steric hindrance, chemical modifications of the amino acid residues within the epitopes and by alteration of the secondary and tertiary structure in proteins (Rait et al., 24b). The study, which used bovine pancreatic RNase, was however conducted using polyclonal antibodies known to interact with more epitopes. In another study, (Rait et al., 24a) show that in formalin fixed tissues, protein enzymes form intra- and inter-molecular crosslinks (Hayat, 21) and attribute these molecular forces among other things to the success of AR. Although there have been attempts by chemical companies to develop varieties of formaldehyde with wide-ranging qualities, a 1985 review article by (Fox et al., 1985) decried the little attention the mechanisms of action of formaldehyde-based-fixatives had received in research, saying it was not matched by its ubiquitous use. Two decades later, lack of further improvement in this area regarding fixation still continues (Yamashita, 27). This perhaps compounded by the fact that during the same period, IHC has been enhanced by the development of techniques in antigen retrieval, a technique that increases immunoreactivity of some antigens following fixation in formalin based fixatives. Alcohol Fixation Alcohols are dehydrants which are used in fixation and in tissue processing. Although used as fixatives, its application for routine purposes is limited due to shrinkage of tissues as well as drying and hardening of tissue blocks. Nuclear patterns also are poorly preserved reducing its value in diagnostic pathology (Grizzle et al., 1998). The 8

20 mechanism of fixation by alcohols is through dehydration and coagulation (Islam Eltoum et al., 21b). Examples of dehydrants in use as fixatives include ethanol, methanol and acetone. Absolute ethanol or methanol are said to be excellent in preserving high molecular weight DNA and RNA (Srinivasan et al., 22). Conversely, (Islam Eltoum et al., 21b) suggest that fixation in dehydrant fixatives cause extraction of lipids that destroys cellular ultra-structure as well as causing shrinkage. (Arnold et al., 1996) in a study of the effects of fixation and tissue processing on immunohistochemical demonstration of specific antigens report that 95% ethanol, absolute methanol and alcoholic formalin give strong immunostaining for p53 and cytokeratins (AE1/AE3), while formalin in a combination with ethanol has been shown to fix carbohydrates in tissues. In routine paraffin section preparations, a 7% ethanol is the first concentration in which tissues are immersed during tissue processing. This concentration known for its bactericidal properties is a good compromise; not too high to cause considerable cytoplasmic shrinkage or distortion of nuclear detail yet not low enough to result in significant swelling of tissue. 7% ethanol however will not remove free water from tissues. 9

21 TISSUE PROCESSING Tissue processing is the next step after fixation in the production of paraffin blocks. The paraffin is an embedding medium that hold tissues to enable cutting thin sections of appropriate consistency and thinness to be mounted on microscope slides before staining for microscopic evaluation. Paraffin is insoluble in water but soluble in organic solvent such as xylene. Xylene on the other hand is insoluble in water. The sequential steps in tissue processing are designed to remove water and establish a hydrophobic environment in order to aid infiltration of paraffin into tissue. This creates a medium that, when the paraffin solidifies, allows for cutting of uniform sections. Dehydration involves removing free water by taking the tissue/cells through a series of increasing concentrations of alcohol, usually ethanol. Here, water and unreactive formaldehyde in the tissues/cells are removed by (alcohol) ethanol, the dehydrant. In clearing, an organic solvent such as xylene is used to remove ethanol from the tissue/cells and infiltrate the tissue creating a hydrophobic environment. This step prepares the tissue/cell for the impregnation by paraffin which is insoluble in water, but soluble in xylene. Clearing also raises the refractive index of the tissue, improving contrast. During impregnation hot paraffin is dissolved in xylene, and the xylene evaporates from the paraffin matrix that now infiltrates tissues/cells. 1

22 Today, tissue processing is performed on instruments designed to optimally infiltrate tissue with paraffin. Very little is known about tissue processing as an entity and its effects on histochemical or immunohistochemical staining (Clive R. Taylor et al., 26; Grizzle et al., 21). Although the effects of such steps as dehydration in alcohol (ethanol) on IHC are known, there s little literature about the effects of clearing and impregnation (Grizzle et al., 21). The closest study about the effects fixation and tissue processing on IHC, by (Werner et al., 2), only addressed problems in delayed fixation and the resulting variation due to the duration of fixation. They suggested that tissue processing has less influence on IHC than fixation. With the advent of methods that can be used to classify tumors molecularly, there is a great need for consistency in tissue fixation and processing. This is very important in that molecular classification based on paraffin blocks is the basis for most archival tissues used in retrospective studies. 11

23 DETECTION SYSTEMS Antibodies on their own attached to antigens are not visible not even when viewed with a light or electron microscope. For the localized antigen to be demonstrated for visualization in a light microscope, there have been several methods employed. These include use of enzymes, fluorescent dyes, colloidal gold and biotin. Enzymes are the most commonly used substances to demonstrated antigen-antibody reactions. Enzymes in the presence of an appropriate substrate and a chromogen will result into a colored precipitate which can be visualized in a light microscope. An example of such a chromogen is 3, 3 diaminobenzedine (DAB). There are two detection systems; direct and the indirect detection system. In the direct detection system the primary antibody is conjugated to a label (reporter molecule) and attached directly to the antigen of interest in the tissue. This method is rapid; however it is less sensitive, requires more antibody and is therefore less economical. In a bid to increase the sensitivity of detection of signal of the antigen, the indirect method was developed. This involved attaching an unlabeled primary antibody to the antigen on the tissue section, and to the primary antibody a labeled secondary antibody. The secondary antibody is a conjugated antibody raised against the antibodies of the species of the primary antibody is derived. Examples of the indirect detection systems include avidin-biotin, peroxide anti peroxidase (PAP), alkaline phosphatase anti alkaline phosphatase (APAAP) methods. The most common of these is the avidin- biotin method. 12

24 Avidin is a high molecular weight glycoprotein found in egg white and has a high affinity for biotin a low molecular weight vitamin. Avidin has four sites available for attachment to biotin. At the same time biotin can also be attached through other sites.to an antibody (biotinylated antibody). Thus, it has a very high binding constant to the biotin on the secondary and this is made use of in detection of antibody. This avidin biotin system is often referred to as ABC; avidin biotin complex. However one disadvantage of the ABC system is the possibility of producing high background, by avidin binding to lectins in tissues through electrostatic binding. Labeled streptavidin techniques, the method used in this study, are an improvement of the ABC method. Streptavidin is extracted from bacteria Streptomyces avidinii is not glcosylated, resulting into a marked reduction of electrostatic binding with tissues thus background staining is less likely. Use of labeled streptavidin techniques shows less background staining due to the less affinity of binding to endogenous biotin. When the label, horseradish peroxidase (HRP) conjugated streptavidin, is applied to the tissue after the secondary antibody an antigen--primary antibody biotinylated secondary--streptavidin HRP complex is formed. The result of this is that HRP enzymes are bound to the tissues at the sites of the antigen of interest. The most common chromogen used is DAB (3,3 -diaminobenzidine) which, in the presence of H2O2 and the HRP enzyme, forms a brown precipitate at the site of the complex. This brown precipitate is easily visible with a light microscope. This method increases sensitivity. Although this method of detection increases signal, it is prone to background staining due to endogenous biotin. Tissues like liver and kidney with high endogenous biotin content 13

25 result in background using this method unless unlabelled avidin and biotin are applied sequentially prior to the primary to block endogenous biotin. 14

26 ANTIGEN RECOVERY Antigen recovery usually applies to any method used to recover antigens (epitopes) following tissue fixation to processing. Antigen retrieval (AR) however was originally used to describe the use of moist heat to increase the staining intensity in archival formalin fixed paraffin embedded tissue (S. R. Shi et al., 21). In the early 199s, Shi et al (S.-R. Shi et al., 1991) observed that the staining intensity of formalin fixed tissues was increased for many antibodies when they used microwave heating in metal solutions. Since then antigen retrieval has been described by several other terms including; antigen unmasking, epitope recovery and epitope retrieval. (Hayat, 21) proposed that antigen retrieval is the best term because it referred to all the parts of a molecule to which the antibody attaches. This is in contrast to epitope which is one antigenic determinant analogous to a paratope on the antibody. He explains that an antigen may have several epitope sites. Currently there is agreement in the use of antigen retrieval as shown by a PubMed search comparing AR with other terms. Although not suited for all for epitopes, heat is the most used form of antigen retrieval method. The effectiveness of heat induced antigen recovery is a result of several factors including the temperature of heating, duration of the heating process and ph of the retrieval solution. Perhaps the blame for lack of active research on the improvement of fixatives (Taylor, 26; Yamashita, 27) should be placed on AR. The prominence of heat as a retrieval method lies in simplicity and ease of performing it. Simple because the 15

27 equipment for use is readily available and does not require complicated manipulation. In performing AR, the objective is to heat the tissue sections or a cell to a condition at which the barriers to epitope recognition are partially or completely destroyed or to a state in which immunostaining is more efficient. Although AR is mainly associated with formalin fixed paraffin embedded tissues, our results indicate that fixation of cells in ethanol also results in masking some epitopes and staining intensity is also increased as a result of AR. The detection of the expression of keratin in ethanol fixed tissue has been reported to be stronger when compared with formalin fixed tissues. Therefore the AR is affected by the type and nature of tissue, type of fixative and method of fixation, ph of the antigen retrieval solution, temperature and time or duration of the retrieval, concentration as well as the clone or type of antibody to be used. However AR alone is inadequate in terms of obtaining satisfactory results from most antibodies (Taylor, 26). In general it is thought that the application of AR reverses the effects of crosslinking by formalin and similar fixatives so that antigens or epitopes of the antigen can be reached and bound by the antibodies. The use of moist heat in antigen retrieval solution is thought effective in unmasking the epitopes, thus optimizing the detection of the antigen (Rait et al., 24b). There have been several methods suggested to conduct AR since (S.-R. Shi et al., 1991) reported its ability to improve immunostaining. Examples used are pressure cookers, microwaves, steamers, kettles, incubators and autoclaves. (Namimatsu et al., 25) have shown on types several tissues that the use of.5% citraconic anhydride, ph 7.4 at 98 C for 45 minutes enhances specific staining. By using a number of antibodies, including some less sensitive ones as well as various tissues to support their study, they deal with a combination which appears to be one of 16

28 the steps that may answer challenges faced in variability in IHC staining. (Evers et al., 1998) while testing two antibodies MAP-2 and SMI-32 on human brain tissue, report that optimal retrieval even when the retrieval solutions were of low ph values; ph 4.5 for MAP-2, and ph 2.5 for SMI-32. (Evers et al., 1998) recommend the use of tris buffered saline of ph for tissue fixed in formalin for several years. Other methods other of antigens recovery include use of enzymes and detergents. Factors to consider use of enzymes, also referred to as proteolytic enzyme induced epitope retrieval, include the concentration of the enzyme, and incubation time. The maximum activity of many enzymes is at about body temperature (37 C), but there is greater control over the digestion process at room temperature. While detergents such as Triton X 1 are used permeabilize cells to facilitate antibody entry, (Hayat, 21) suggests that the role of such reagents ought to be included among factors that affect AR since they are sometimes used in combination with heating. 17

29 METHODOLOGY To have a clear insight into the effects of duration in fixative on epitope masking, DU145 and SKOV3 cells grown on microscope slides were used as models of fresh tissues. In experimental design I, using DU145 cells, we chose six times of fixation to compare: short (no fixation (m), 5m and 6h), moderate (24h) and long (48h, 72h). These fixation times were combined with the cumulative steps of tissue processing to identify if immunostaining was affected differently by the various times of fixation depending upon the stages up to tissue processing. For example in order to mimic the first step in tissue processing, after all the microscope slides had been fixed for various times (no fixation (m), 5m, 6h, 24h, 48h and 72h), all the were then incubated at the same time in 7% ethanol for 1h; this modeled the first non-fixative step on a tissue processor. Then, in the same run, all slides were immunostained described subsequently. Experimental design II using SKOV3 cells, utilized a similar approach based on DU145 results, except only four fixation time points were considered as basis for generalization: i.e., short (no fixation (m), 5m and 6h) and moderate (24h). Also, with the benefit of understanding of the response by DU145 cells, only four key cumulative stages of tissue processing were evaluated i.e.:- 1. Fixation in 1% at the four times alone. 2. Fixation at the four times together with 7% ethanol for 1h. 18

30 3. Fixation at the four times together with 7% ethanol for 1h, 8% ethanol for 1h, 9% ethanol for 1h plus absolute ethanol for 3h representing dehydration and finally, 4. Fixation with each of the four time points of fixation plus dehydration from 7% ethanol through to absolute ethanol for 3h plus 2h of exposure to xylene. In experimental designs I and II all conditions needed to be stained at the same time so the slides requiring longer durations of fixation were incubated earlier in 1% to allow all times of fixation to converge; with the goal of staining all slides representing a cumulative point on the tissue processor at the same time. Practically, this required each step of the tissue processor to be modeled in a separate experimental run; therefore, because staining was performed on different days, upon completion of the entire experiment we had a total of six staining runs with DU145 cells and four for SKOV3 cells. In order to rule out variations in the extent of staining during the individual staining, experimental design III was desired. Experimental design III utilized one time of fixation (24h - the moderate time) and only DU145 cells. It was planned in such a way that each of the tissue processing steps after 24h of fixation could be compared. Also, the individual cumulative tissue processing steps were run to as seven experiments converge at the same time for immunohistochemical staining. This permitted a direct comparison of the effect of each of the combined stages of tissue processing; thus:- a. the absence of tissue processing could be compared, b. with 1h in 7% ethanol which could be compared, c. with 1h in 7% ethanol and 1h in 8% ethanol, 19

31 d. with 1h in 7% ethanol, 1h in 8% ethanol plus 1h in 9% ethanol, e. with 1h in 7%, 1h in 8%, 1h in 9% and 3h in absolute ethanols, f. with 1h in 7%,1h in 8%, 1h in 9%, 3h in absolute ethanols plus 2h in xylenes and finally, g. with 1h in 7%, 1h in 8%, 1h in 9%, 3h in absolute ethanols, 2h in xylene plus 2h in paraffin. Essentially experimental design I, II and III are similar; with experimental designs I and II permitting the comparison of the times of fixation at the cumulative stages of tissue processing and experimental design III permitting the comparison of effects on immunostaining of the cumulative steps in tissue processing at one specific time of fixation. In all experimental designs the different responses of immunostaining to fixation or to tissue processing steps were evaluated by estimation of the intensity of cellular nuclear staining with one of the two anti-bodies; Ki67/MIB-1 chosen because it requires antigen recovery and PCNA which does not require antigen recovery for IHC. Slide Preparation: Pre-cleaned microscope slides Surgipath #299 (525 Rt. 12 Richmond, IL) were marked/ identified with information including; the antibodies and their concentration, cell line and time points. The slides were immersed in a staining dish of 1% poly-lysine (Sigma Aldrich Inc, St. Louis, MO) for 5m, removed and left to dry over night at room temperature. The coated slides were next transferred in Petri dishes (Pyrex 14cm diameter) and autoclaved. This treatment considerably reduced the variations in 2

32 staining of cells observed earlier in initial experiments using other types of slides. Variable staining was thought secondary to irregular coating of slides by vendor methods of slide preparation. Cellular Preparation: Two cell lines, DU145 (ATCC Manassas, VA) and SKOV3 (ATCC Manassas, VA) were used so that results could be generalized and were not specific for just one cell line. The cells were cultured using ATCC recommended guidelines and media, harvested by trypsinization and the cell-suspension seeded under biological safety cabinet on to preautoclaved Petri-dishes that contained sterile microscope slides coated with poly-lysine. The cells were then incubated in a water jacketed incubator (Forma Scientific, Inc.) at 5% CO2 atmosphere at 37 C and on the following day (for DU145), the attached cells on microscope slides were rinsed in PBS and fixed in 1% (Richard Allan Scientific, Kalamazoo, MI) for different times (i.e., no fixation (m), 5m, 6h, 24h, 48h and 72h) for DU145 and (no fixation (m), 5m, 6h and 24h for SKOV3). To allow for the different experimental time points to converge and enable same time staining in one run, microscope slides were stored in a refrigerator at 4 C (e.g., the 72h slides were incubated in 1% at 4 C for the 72h duration of fixation). Each time of fixation was combined with one of the cumulative steps in tissue processing for each staining run in experimental design I specifically; no fixation (m), 5m, 6h, 24h, 48h and 72h would be combined with 1h in 7% ethanol in one immunostaining run while these same times of fixation would be combined with the one cumulative steps in tissue processing 21

33 1h in 8%, 1h in 9%, 3h in absolute ethanol, 2h in xylene, and 2h in paraffin in separate staining runs. Experimental approach I Compare each fixation time m, 5m, 6h, 24h, 48h and 72h combined with each of the cumulative conditions of tissue processing in a separate staining run (1st staining run). a. 1h 7% ethanol (2nd staining run). b. 1h 7%, 1h 8% plus 1h in 9% ethanols (3rd staining run). c. 1h 7%, 1h 8%, 1h 9% plus 3h absolute ethanols (4th staining run). d. 1h 7%, 1h 8%, 1h 9%, 3h absolute ethanols plus 2h of xylene (5th staining run). e. 1h 7%, 1h 8%, 1h 9%, 3h absolute ethanols plus infiltrate with wax (paraffin) (6th staining run). In experimental design III, DU145 cells were fixed only for 24h and the cells attached to microscope slides were rinsed with PBS and combined with either no tissue processing or one of the cumulative steps of tissue processing to ensure staining took place in the same run. Experimental approach II Compare 24h of fixation together with each of the following; a. Nothing. b. 1h 7% ethanol. c. 1h 7% plus 1h 8% ethanol. d. 1h 7%, 1h 8% plus 1h 9% ethanols. 22

34 e. 1h 7%, 1h 8%, 1h 9% plus 3h in absolute ethanols. f. 1h 7%, 1h 8%, 1h 9%, 3h in absolute ethanols plus 2h in xylene. g. 1h 7%, 1h 8%, 1h 9%, 3h in absolute ethanols, 2h in xylene plus 2h in wax (paraffin). Then each condition a) to g) is immunostained together in the same staining run. This permits the evaluation of the cumulative stages of tissue processing. Immunostaining and the rest of the procedures was the same as for experimental designs I and II. Immunostaining: To minimize possible changes in immunohistochemical staining due to variations in staining (Grizzle et al., 1998), in experimental approach I all slides for one cell line were stained on the same day. Specifically microscope slides were rinsed in Tris buffer ph 7.6 for 1m, and permeabilized (membrane perforation) in.5% Triton X-1 (Sigma Aldrich Inc, St. Louis, MO) in PBS for 3m. The AR slides were submerged in plastic Coplin jars filled in.1m EDTA ph 8 in an electric pressure cooker (Cook s essentials model CEPC 8) containing approximately 5mls of DI water. Heating time for antigen recovery was 5m. PAP pens were used to mark the microscope slides so that drainage of reagents was minimized; the slides were loaded on to a DAKO Autostainer programmed to the standard protocol used in the laboratory. The Autostainer has flexible programming set so that the slides undergo 5m of exposure to 3% aqueous H2O2 to quench endogenous peroxidase, 1h of 3% goat serum block to reduce nonspecific staining, and 1h of incubation with each primary antibody. The primary antibodies were Ki67/MIB-1 (Biogenex, San Ramon, CA) 1:12 or PCNA (Santa Cruz Biotechnology, 23

35 Santa Cruz, CA) 1:18, both diluted in PBE ph 7.6. For controls 3% goat serum was used to replace the primary antibody as a delete. All slides were rinsed with Tris buffer ph 7.6 and incubated 1m in a multispecies of Link (Signet, Dedham, MA) washed, incubated 5m in Streptavidin Label (Signet, Dedham, MA) and washed before switching to incubation for 7m with 3,3 -diaminobenzedine (DAB) (Biogenex, San Ramon, CA) to produce an insoluble chromogen. The slides finally were rinsed with DI water, counter stained in Harris hematoxylin for 1m 15s, blued in tap water, dehydrated in 7%, 95% and absolute ethanol, before clearing in three changes in xylene ( Fisher Scientific, Fair Lawn, NJ) (5m each from dehydration to clearing). Coverslips were mounted on the slides using permount as a mountant. The slides were stored at room temperature until evaluated. Effects of Tissue Processing: In the next of this series of experimental design I, the prior procedure was followed as described previously, i.e., slide preparation, cell culturing (multiplication and seeding) and fixation for (no fixation (m), 5m, 6h, 24h, 48h and 72h). To mimic the first dehydration step in tissue processing, without rinsing, all slides were immediately incubated in 7% ethanol for 1h following a similar procedure of IHC as in the previous fixation only experiment. In this batch, no fixation (m) slides now were incubated in 7% ethanol for 1h. In the third staining run of experimental design I, like prior experiments, cells were cultured on prepared slides, fixed at the various times and, then without rinsing, incubated in staining dishes at increasing concentrations of ethanol - 7% ethanol for 1h, 24

36 8% ethanol for 1h, and 9% ethanol for 1h then stained for immunohistochemical evaluation. In experimental design I, the 8% step was combined with the 9% step because we did not expect a significant change between fixation and tissue processing in 7% ethanol for 1h and fixation and tissue processing for 1h in 8% ethanol and 1h in 9% ethanol; however the 1h 8% ethanol step was included in experimental design III which was designed to compare the cumulative steps of the tissue processing. In the fourth staining run of experimental design I, following cell culture and fixation, the slides were incubated in staining dishes with increasing concentrations of ethanol as follows; 7% ethanol for 1h, 8% ethanol for 1h, 9% ethanol for 1h and absolute ethanol for 3h and then the cells were stained by IHC. The fifth staining run of experimental design I included all the previous cumulative stages of tissue processing plus incubation of the cells on slides in xylene for 2h before immunohistochemical staining. In the last staining run of experimental design I, all the previous cumulative steps were followed, but in this experiment the cells were incubated in a paraffin (Ameraffin LP, Cardinal Health, McGaw Park, IL) bath maintained at 62 C for 2h. For only this staining run, before staining for IHC, the cells on the slides were deparaffinized in three changes of xylene 5m each, rehydrated in a series of decreasing concentrations of ethanol absolute ethanol, 95% ethanol and 7% ethanol 5m at each concentration, immediately transferred in Tris buffer ph 7.6 for 1m and then the usual immunohistochemical procedure was followed. 25

37 Summary of Tissue Processing: Experimental design I: In summary, all times of fixation in 1% (i.e. 5m, 6h, 24h, 48h and 72h) were followed by the cumulative steps in tissue processing. Included in the step of tissue processing were slides with no fixation in. RUN 1: RUN 2: RUN 3: No tissue processing (fixation alone) immunostaining. 1h in 7% ethanol followed by immunostaining. 1h in 7% ethanol, plus 1h in 8% ethanol plus 1h in 9% ethanol followed by immunostaining. RUN 4: 1h in 7% ethanol, plus 1h in 8% ethanol, plus 1h in 9% ethanol, 3h in absolute ethanol followed by immunostaining. RUN 5: 1h in 7% ethanol, plus 1h in 8% ethanol, plus 1h in 9% ethanol, plus 3h in absolute ethanol, plus 2h in xylene followed by immunostaining. RUN 6: 1h in 7% ethanol, plus 1h in 8% ethanol, plus 1h in 9% ethanol, 3h in absolute ethanol, plus 2h in xylene, plus 2h in paraffin followed by immunostaining. Prior to immunostaining, cells on microscope slides were processed either through part (e.g. run 4) or all (i.e., run 6) of the steps in tissue processing. For simplicity, the 8% and 9% steps were combined in run 4. Immunostaining of runs 1-6 were on separate days. In experimental design III, fixation for 24h was followed by processing following a reverse schedule of tissue processing to ensure that all staining was done at the same time/run. Fixation is 24h prior and coincides with start of processing. STEP 1 Start processing through paraffin 8:AM STEP 2 Start processing through xylene 1:AM 26

38 STEP 3 Start processing to absolute ethanol 11:AM STEP 4 Start processing to 9% ethanol 2:PM STEP 5 Start processing to 8% ethanol 3:PM STEP 6 Start processing to 7% ethanol 4:PM STEP 7 paraffin slides are deparaffinized, xylene is removed and slides are hydrated; fixation of 24h for the no processing group ends and immunostaining of all slides begins. 5:PM Steps 1-7 all take place on the same day Analysis Five fields of stained the stained slides were photographed at magnification of X4 using a Nikon model 14. microscope (connected to a computer Hitachi SuperScan Pro 8) camera and the pictures of each field were printed separately using Hp desk jet 99CSE professional series. In order to assess fully the effect of duration of fixation and tissue processing, a scoring system previously developed in our laboratory(arnold et al., 1996) was used; it was based on the intensity of nuclear staining in which nuclei were categorized from. for no nuclear staining up to a maximum of 4. for darkest staining. The range between no nuclear staining to darkest was evaluated in.5 increments with the progressive increase in staining as follows: (.,.5, 1., 1.5, 2., 2.5, 3., 3.5, and 4.). At a fixed Ki67/MIB-1 and PCNA 1 concentrations, the changes in staining over time after 1% and separately the individual steps of tissue processing was evaluated. 27