The Effect of Formaldehyde on Tissue Lipids and on Histochemical Reactions for Carbonyl Groups

Histcchemical and chemical evidence indicates that formaldehyde combines with unsaturated lipids at the double bond. The resulting complex contains a free carbonyl group which probably originates from the formaldehyde. The reaction occurs over a wide pH range, and takes place in the absence of oxygen or moisture. The reaction product is visualized by the Schiff reagent, and by the Ashbel-Seligman procedure. In the plasmal procedure, when performed on formalin-treated material, the reaction has the same significance as the pseudo-plasmal reaction, i.e. it denotes the presence of double bonds. The Ashbel-Seligman technic seems to be more sensitive to this complex than the Schiff reagent and shows it more markedly than it does the true plasmals and the atmospherically oxidized unsaturated compounds.     

Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links

Abstract

The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.     

Combined effects of formalin fixation and tissue processing on immunorecognition

Abstract

It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.     

Cedarwood Oil as a Clearing Agent with Acetic-Alcohol Fixatives

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3–24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehydecontaining solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.     

News in Brief

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.     

Development Of A Nucleic Acid Lateral Flow Strip For Detection Of Hepatitis C Virus (Hcv) Core Antigen

The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10⁴ copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

Proteomics of human prostate cancer biospecimens: the global,systems-wide perspective for Protein markers with potential clinical utility

Despite intense global efforts, no new clinical and/or viable biomarkers have been established to overcome the limitation of the prostate specific antigen in the early diagnosis and prognosis of prostate cancer (PCa). The current proteomic approaches to PCa biomarker discovery, each have distinct advantages and disadvantages, yet when combined hold real promise in the coming years. One key approach to this effort is the development of non-targeted, depletion-free and quantitative liquid chromatography–ultra high resolution tandem mass spectrometry (LC–MS) pipelines for the systems-wide interrogation of the diverse proteomes encompassed in whole tissue and blood serum or plasma. Derived quantitative proteomes can be decoded for their biomedical relevance with advanced bioinformatics and bibliographic mining to yield promising ‘molecular portraits’ that can gauge prostatic disease at the serological level. Their functional annotation, although potentially useful, is beyond our current level of biological understanding and should not be requisite for their effective use in the clinical monitoring of prostatic disease.     

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a “buffering” effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0–7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.     

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells^3-5. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells⁶, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies¹. Originally published by Naal et al.¹, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease^7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function². In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε₂₈₀ = 4,200 L/M/cm)¹². This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.