TdTomato and EGFP identification in histological sections: insight and alternatives

Abstract

Tandem dimer Tomato (tdTomato) provides a useful alternative to enhanced green fluorescent protein (eGFP) for performing simultaneous detection of fluorescent protein in histological sections together with fluorescence immunohistochemistry (IHC). eGFP has many properties that make it useful for cell labeling; however, during simultaneous fluorescence IHC, the usefulness of eGFP may be limited. This limitation results from a fixation step required to identify eGFP in histological tissue sections that can mask antibody epitopes and adversely affect staining intensity. An alternative fluorescent protein, tdTomato, may assist concurrent detection of fluorescent protein within tissue sections and fluorescence IHC, because detection of tdTomato does not require tissue fixation. Tissue sections were obtained from various organs of mice ubiquitously expressing eGFP or tdTomato that were either unfixed or fixed with 4% paraformaldehyde. These tissues later were combined with fluorescence IHC. Both eGFP and tdTomato displayed robust signals in fixed frozen sections. Only tdTomato fluorescence, however, was detected in unfixed frozen sections. Simultaneous detection of fluorescence IHC and fluorescent protein in histological sections was observed only in unfixed frozen tdTomato tissue. For this reason, tdTomato is a useful substitute for eGFP for cell labeling when simultaneous fluorescence IHC is required.     

Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA.

The standard method for assessment of cell proliferation in paraffin-embedded tissue sections is 5-bromodeoxyuridine (BrdU) immunohistochemistry (IHC). BrdU can be administered to laboratory animals via IP injections, is readily incorporated into nuclei during the DNA synthetic phase of the cell cycle, and is detected with an anti-BrdU antibody. This method has several disadvantages, and an accurate method for evaluation of proliferative activity that can substitute for BrdU IHC, when necessary, is of great interest to investigators. Alternative methods for detection of proliferating cells in tissue sections are proliferating cell nuclear antigen (PCNA) IHC, Ki-67 IHC, and in situ hybridization (ISH) for histone mRNA. To determine the optimal choice, we analyzed the correlation of anti-PCNA, anti-Ki-67(MIB-5), and histone mRNA labeling indices (LIs) with anti-BrdU LI in rat highly replicative (renewing) tissues. The correlation between anti-BrdU and histone mRNA LIs, as well as the correlation between anti-BrdU and anti-Ki-67 LIs, was statistically significant. There was no significant correlation between anti-BrdU and anti-PCNA LIs. These results suggest that both ISH for histone mRNA and IHC with MIB-5 are preferable techniques for assessment of cell proliferation in rat paraffin-embedded renewing tissues compared to PCNA IHC. They can substitute for BrdU IHC when necessary.     

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)     

Development of multigene expression signature maps at the protein level from digitized immunohistochemistry slides

Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed,paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Synergistic Tissue Counterstaining and Image Segmentation Techniques for Accurate, Quantitative Immunohistochemistry

Quantitative analysis of digitized IHC-stained tissue sections is increasingly used in research studies and clinical practice. Accurate quantification of IHC staining, however, is often complicated by conventional tissue counterstains caused by the color convolution of the IHC chromogen and the counterstain. To overcome this issue, we implemented a new counterstain, Acid Blue 129, which provides homogeneous tissue background staining. Furthermore, we combined this counterstaining technique with a simple, robust, fully automated image segmentation algorithm, which takes advantage of the high degree of color separation between the 3-amino-9-ethyl-carbazole (AEC) chromogen and the Acid Blue 129 counterstain. Rigorous validation of the automated technique against manual segmentation data, using Ki-67 IHC sections from rat C6 glioma and β-amyloid IHC sections from transgenic mice with amyloid precursor protein (APP) mutations, has shown the automated method to produce highly accurate results compared with ground truth estimates based on the manually segmented images. The synergistic combination of the novel tissue counterstaining and image segmentation techniques described in this study will allow for accurate, reproducible, and efficient quantitative IHC studies for a wide range of antibodies and tissues. (J Histochem Cytochem 56:873–880, 2008)     

Identification of 5-Bromo-2’-Deoxyuridine-Labeled Cells during Mouse Spermatogenesis by Heat-Induced Antigen Retrieval in Lectin Staining and Immunohistochemistry

DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2’-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes.     

No detection of XMRV in blood samples and tissue sections from prostate cancer patients in Northern Europe

Background

We recently published the rare detection of xenotropic murine leukemia virus-related virus (XMRV) (1/105) in prostate cancer (PCA) tissue of patients in Northern Europe by PCR. The controversial discussion about the virus being detected in PCA tissue, blood samples from patients suffering from chronic fatigue syndrome (CFS), as well as from a significant number of healthy controls prompted us to deepen our studies about detection of XMRV infection applying different detection methods (PCR, cocultivation and immunohistochemistry [IHC]).

Methodology/Principal Findings

Peripheral blood mononuclear cells (PBMCs) from 92 PCA and 7 healthy controls were isolated, PHA activated and cocultivated with LNCaP cells for up to 8 weeks. Supernatant of these cells was applied to a reporter cell line, DERSE-iGFP. Furthermore, the PBMCs and cocultivated LNCaP cells were tested for the presence of XMRV by PCR as well as Western Blot analysis. While all PCR amplifications and Western Blot analyses were negative for signs of XMRV infection, DERSE-iGFP cells displayed isolated GFP positive cells in three cases. In all three cases XMRV presence could not be confirmed by PCR technology. In addition, we performed XMRV specific IHC on PCA tissue sections. Whole tissue sections (n = 20), as well as tissue microarrays (TMA) including 50 benign prostate hyperplasia (BPH), 50 low grade and 50 high grade PCA sections and TMAs including breast cancer, colon cancer and normal tissues were stained with two XMRV specific antisera. XMRV protein expression was not detected in any cancer sections included. One BPH tissue displayed XMRV specific protein expression in random isolated basal cells.

Conclusion

We were unable to conclusively detect XMRV in the blood from PCA patients or from healthy controls and there is no conclusive evidence of XMRV protein expression in PCA, breast cancer and colon cancer tissue sections tested by IHC staining.     

Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis.

From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. "The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue". This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of integral(Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as integral (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = integral (Pffpe)/ integral (Pf) x 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf x R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe= Pf. Further studies are designed to test the limitations of the proposed hypothesis.     

IHC Profiler: An Open Source Plugin for the Quantitative Evaluation and Automated Scoring of Immunohistochemistry Images of Human Tissue Samples

In anatomic pathology, immunohistochemistry (IHC) serves as a diagnostic and prognostic method for identification of disease markers in tissue samples that directly influences classification and grading the disease, influencing patient management. However, till today over most of the world, pathological analysis of tissue samples remained a time-consuming and subjective procedure, wherein the intensity of antibody staining is manually judged and thus scoring decision is directly influenced by visual bias. This instigated us to design a simple method of automated digital IHC image analysis algorithm for an unbiased, quantitative assessment of antibody staining intensity in tissue sections. As a first step, we adopted the spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. Then the DAB stained image is displayed in a new window wherein it undergoes pixel-by-pixel analysis, and displays the full profile along with its scoring decision. Based on the mathematical formula conceptualized, the algorithm is thoroughly tested by analyzing scores assigned to thousands (n = 1703) of DAB stained IHC images including sample images taken from human protein atlas web resource. The IHC Profiler plugin developed is compatible with the open resource digital image analysis software, ImageJ, which creates a pixel-by-pixel analysis profile of a digital IHC image and further assigns a score in a four tier system. A comparison study between manual pathological analysis and IHC Profiler resolved in a match of 88.6% (P<0.0001, CI = 95%). This new tool developed for clinical histopathological sample analysis can be adopted globally for scoring most protein targets where the marker protein expression is of cytoplasmic and/or nuclear type. We foresee that this method will minimize the problem of inter-observer variations across labs and further help in worldwide patient stratification potentially benefitting various multinational clinical trial initiatives.     

A natural case of chronic wasting disease in a free-ranging moose (Alces alces shirasi)

Chronic wasting disease (CWD) was diagnosed in a free-ranging moose (Alces alces shirasi) killed by a hunter in Jackson County, Colorado, USA, in September 2005. The diagnosis was based upon immunohistochemistry (IHC) demonstrating the presence of accumulations of CWD-associated prion protein (PrP(CWD)) in tissue sections of medulla oblongata at the level of the obex (dorsal motor nucleus of the vagus) and in retropharyngeal lymph node (RPLN); additional testing by IHC revealed deposits of PrP(CWD) in multiple sections of medulla oblongata and cervical spinal cord as well as palatine tonsil and submandibular lymph node tissues. Western blot confirmed the presence of PrP(CWD) in RPLN and tonsil tissue. The PrP(CWD) also was detected via enzyme-linked immunosorbent assay of RPLN tissue. Spongiform encephalopathy was observed in sections of the brainstem and cervical spinal cord, although no clinical signs were noted by the hunter who killed the animal. The affected moose was homozygous for methionine at codon 209 of the prion protein coding region. In October 2006, two additional free-ranging moose were diagnosed with CWD. Epidemiology and implications of CWD in moose remain to be determined.     

Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report

Intratumoral aromatase is a potential therapeutic target for the treatment of postmenopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. A multi-center collaborative group has been established to generate and validate new aromatase monoclonal antibodies (MAbs). A recombinant GST–aromatase fusion protein was expressed in baculovirus and the purified protein was used for immunization of mice either as a native or formalin-fixed antigen. Hybridomas were generated using standard techniques and screened biochemically prior to immunohistochemistry (IHC) evaluation in human placenta, ovary and breast cancer tissues. Twenty-three MAbs selected by biochemical assays were further evaluated by IHC of paraffin-embedded tissue sections including normal ovary, and placenta, and a small series of 10 breast carcinomas. Of the 23 MAbs, 2 (clones 677 and F2) were determined to specifically stain cell types known to express aromatase in normal tissues. In breast carcinomas staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments was detected. IHC was performed and independently evaluated by three pathologists (HS, TJA and SGS), each using the same evaluation criteria for staining intensity and proportion of immunopositive cells. With these two MAbs, interpathologist and intralaboratory variations were minimal in comparison with differences which could be detected between tissue specimens and antibodies.     

Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification.

To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor alpha2 (GFRalpha2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFRalpha2 mRNA and virtually all GFRalpha2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFRalpha2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization.     

Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.