Background Staining Research Articles

P1.01-042 Molecular Epidemiology of Programmed Cell Death 1-Ligand 1 (PD-L1) Protein Expression in Non-Small Cell Lung Cancer

Expression of programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) patients might identify patients who would benefit from PD-L1 blocking antibodies. In a retrospective cohort of NSCLC patients, we characterized PD-L1 expression and other biomarkers to determine if PD-L1 expression is a prognostic biomarker and whether patient characteristics could be identified to determine those associated with high expression. This was a retrospective analysis of 136 NSCLC patients diagnosed between 1997 and 2015 with stage IIIB and IV disease and treated at Moffitt Cancer Center and affiliated institutions. All patients had at least 2 lines of standard of care chemotherapy and sufficient archival tumor tissue for PD-L1 testing by the Ventana SP263 validated assay and mutation status testing by targeted DNA sequencing with the TumorCare Panel. High PD-L1 expression was defined as ≥ 25% of tumor cells with membrane positivity for PD-L1 at any intensity above background staining. Statistical analyses were performed comparing PD-L1 expression by patient characteristics. Survival analyses were performed using Kaplan-Meier survival curves and the log-rank statistic. All statistical tests were two-sided; P-value of less than .05 was considered statistically significant. Of the 136 tissues tested for PD-L1 expression, 116 (85.3%) were collected by surgical resection and 20 (14.7%) were collected by biopsy. Mean sample age was 7.2 years (SD=2.8 years). 82 of the 136 samples also underwent targeted DNA sequencing. In this patient cohort, 51.5% were male, 83.1% were ever smokers, 90.4% were White, 39% were stage IV at time of tissue collection, 71.3% had adenocarcinoma, 28.7% had four or more lines of therapy, and 24.2% had high-expression for PD-L1. There were no statistically significant differences with respect to PD-L1 expression for patient characteristics, overall survival (OS), or progression-free survival (PFS). Additionally, there were no statistically significant differences with respect to PD-L1 expression by EGFR (WT/PD-L1<25% = 74.4% vs. WT/PD-L1≥ 25% = 88.5%), KRAS (WT/PD-L1<25% = 74.7% vs. WT/PD-L1≥ 25% = 69.2%), and ALK status (Neg/PD-L1<25% = 98.5% vs. Neg/PD-L1≥ 25% = 100%). However, mutation load (total number of non-synonymous mutations) was statistically significantly correlated with PD-L1 expression (correlation coefficient = 0.23; P = 0.03). In this study of NSCLC patients treated with 2 or more lines of standard of care chemotherapy, PD-L1 expression (high vs. low and as a continuous covariate) was not statistically significantly associated with OS or PFS. We did observe a novel positive correlation between PD-L1 expression and mutational load.

Vegetable Stain as an Alternative to H&amp;E in Exfoliative Cytology

Abstract Experimentally red beet additionally referred to as beet root (Beta vulgaris L.) used as vegetable stain. Objective: To organize a dye extract from beet root to stain the buccal smear as substitution low cost and simple obtaining material. Material and method: Collected buccal smear from patient in Saveetha dental faculty around ten buccal smears every and glued in alcohol for half-hour. Then the staining was finished beet root extract answer. Results: The cells were stained brown in color with beet root. This study showed that the preservation of cellular morphology was gift and clarity of cellular details was moderate. Additionally showed delicate staining uniformity and background staining was gift. Conclusion: Beet root is accustomed stain buccal smear in exfoliative microscopic anatomy.

Leishman-Giemsa Cocktail - Is it an Effective Stain for Air Dried Cytology Smears.

Air dried cytology smears are stained routinely with Romanowsky stains so that the relative cell size, nuclear size, cytoplasmic details, smear background elements and intercellular matrix components are better appreciated. A variety of modified Romanowsky stains are used in cytology. Leishman-Giemsa (LG) cocktail is one of the new staining techniques which can be used for staining the air dried cytology smears. To evaluate the quality of staining of LG cocktail on air dried smears and to compare the quality of staining of LG cocktail with May Grunwald Giemsa (MGG) which is the most commonly used stain in cytology. The present prospective comparative study was carried out with 100 cases and two extra smears were prepared for each case and stained with MGG and LG cocktail stains. The stained slides were blinded and were evaluated for the staining characteristics of the nucleus, cytoplasm and background staining. Based on this, scoring was done by two pathologists independently. Quality Index (QI) was calculated by dividing the scores obtained with the total score possible. LG cocktail stained slides were excellent in cytoplasmic staining, granularity, nuclear morphology, background material staining and overall staining characteristics. QI of LG cocktail was 0.8 while that of MGG was 0.59. Staining of air dried smears by LG cocktail has a good QI. It is also cheaper, requires short duration for staining as compared to MGG. Hence, LG cocktail can be an effective replacement for MGG for staining the air dried cytology smears.

P1.02-029 Infrequent Staining Patterns in ALK Immunohistochemistry: Correlation with Fish Analysis

AKL gene rearrangements are predictive alterations in non-small cell lung carcinomas (NSCLC). Immunohistochemistry (IHC) has become a valuable tool in assessing ALK status, however unusual staining patterns, such as heterogeneous diffuse moderate and focal intense stains, may occur and can make evaluation difficult. We correlated immunohistochemistry unusual staining patterns with ALK status by fluorescence in situ hybridization (FISH). Of 851 cases tested, we found 14 (1.6%) cases with inconclusive staining patterns that can be summarized in: a) diffuse granular cytoplasmic moderate stain, with or without background mucin stain (10 cases) b) focal intense granular cytoplasmic stain in overall negative or weakly positive tumors (4 cases). IHC was performed on an automatized Benchmark staining module using Ventana ALK (D5F3) CDx assay with Optiview amplification kit. FISH was performed using ALK break-apart probe set (Vysis LSI ALK Dual Color, Abbott Molecular). Cases were considered ALK-FISH positive if ≥15% tumor cells showed split red and green signals (separation of 2 diameters or more) and/or single red signals. Of 10 moderate granular cytoplasmic stain cases, 4 had also abundant mucin background stain 6 where markedly heterogeneous with areas of weak and moderate cytoplasmic granular stain. Nine were FISH negative, one yielded no signals (uninformative result) and one specimen corresponded to an acid decalcified specimen and was not evaluated by FISH. Focal intense stain was observed in 5 samples, 3 corresponded to surgical specimens and the rest to small needle biopsies, one of the surgical specimens was FISH positive and the rest, negative. Since FDA approval of Ventana ALK (D5F3) IHC CDx Assay, IHC has become a widely used tool for assessing ALK status. Guidelines suggest that weak granular stain should be interpreted as negative and focal intense granular stain in any number of cells, as positive. Even though our sample is small, moderate granular stain was consistently negative by FISH analysis, however, focal intense stain shows more discordant results between tests. To date, no suggestions are made on what should be the minimum amount of tumor in a sample to report an IHC assay. Even though some of these patients with IHC positive/FISH negative results have been reported as responders to Crizotinib, further studies are needed. One specimen with moderate cytoplasmic IHC stain was uninformative due to lack of signals. This raises the issue of the need to standardize pre-analytical variables, which can be difficult in some areas of Latin America.

Acetic acid urea-polyacrylamide gel electrophoresis: a rapid method for testing the genetic purity of sunflower seeds

Genetic purity testing is an important component of seed quality control. The objective of this study was to develop a rapid method for testing the genetic purity of sunflower seeds. We used six sunflower hybrids and their parental lines to study and improve a method based on the acetic acid urea-polyacrylamide gel electrophoresis (AU-PAGE) approach for testing the genetic purity of maize seeds. The results showed that the most abundant proteins in sunflower seeds were water-soluble proteins, followed by salt-soluble proteins, while there were relatively few other proteins. The AU-PAGE method was improved to increase the clarity of lanes backgrounds by adding an appropriate amount of sodium dodecyl sulfate to the protein extracting solution. The reduced background staining of the lanes improved the bands identification and increased the evidence of protein polymorphism. The method was validated as an efficient method for genetic purity testing of sunflower seeds. The improvements in this method could potentially be applied to other plants and other protein electrophoresis.

Dual-color chromogen In Situ hybridization (CISH) for the determination of Her2 status in breast cancer tissue sections

Background: A factor associated with breast cancer development is the mutation of the Her2/neu gene located on chromosome 17. Chromogen in situ hybridization (CISH) in which the color is generated with an enzymatic reaction is a promising alternative detection method to fluorescence in situ hybridization (FISH) which is nowadays used in clinical practice. The resulting color is stable and therefore usable for permanent storage, the use of a normal brightfield microscope is possible and the morphology of the sample can be evaluated more easily than with a fluorescence microscope. For the visualization of Her2/neu it is however important to also detect chromosome 17 and use it as a reference chromosome. The aim of the study is: to select the best substrates considering color production, background staining, reactivity and localization. Secondly to develop a dualcolor procedure visualizing Her2/neu and chromosome 17 to examine the possibility of using CISH as a standardized method for the detection of Her2 positive tumors and finally to compare these results with FISH. Methods: Single target CISH with eight substrates for the enzyme horseradish peroxidase (PO) and six substrates for alkaline phosphatase (AP) was performed on cervical cancer cell lines with centromere 1 (C1) and 1p36 as targets. The best substrates were subjectively selected with the use of a brightfield microscope. The procedure was expanded to a dual-color detection on cells and formalin-fixed, paraffin embedded (FFPE) breast cancer tissue sections with Her2/neu and centromere 17 (C17) as targets. In this procedure the best conjugate detection systems were worked out. Results: In the single target detection procedure the substrates DAB, Vina Green and Seramun Grun for PO and Ferangi Blue and Vulcan Fast Red for AP were found to give the best color reactions. The dual-color detection on tissue sections showed strong and good distinguishable color reactions when combining the substrates Vina Green for C17 and Vulcan Fast Red for Her2. Results of CISH and FISH were comparable when using immersion oil for the detection of the fluorescent signals. Discussion and Conclusion: Vina Green for C17 and Vulcan Fast Red for Her2/neu are the best substrates considering background staining, color distinguishability and localization and give comparable results to FISH. The study gives a further step in the direction of introducing CISH as a standardized method for the detection of Her2/neu amplification.

Development and validation of an immunohistochemistry procedure for the detection of a neurotropic bovine astrovirus

Members of the Astroviridae family are best known to cause diarrhea in different mammalian species. Lately, some strains have been associated with encephalitis in humans, minks and cattle. In this study, we developed an immunohistochemistry (IHC) procedure for the detection of a neurotropic bovine astrovirus (BoAstV-CH13/NeuroS1), which is associated with non-suppurative encephalitis in cattle. We expressed five recombinant antigens corresponding to different putative viral proteins of BoAstV-CH13/NeuroS1. Antigens were then used for the production of hyperimmune sera in rabbits. Out of the five hyperimmune sera, the one directed against the conserved N-terminus of the viral capsid protein, termed ORF2-con, clearly surpassed the others in the detection of viral antigens in IHC in terms of strong signal intensity and low background staining. The accuracy of the ORF2-con IHC protocol was then evaluated using different sets of brain tissue samples: 30 samples from 9 animals with confirmed BoAstV-CH13/NeuroS1 infection, 30 samples from 8 animals with non-suppurative encephalitis of another etiology and 30 samples from apparently healthy slaughtered animals. The IHC was positive only with tissue samples from animals with a known positive BoAstV-CH13/NeuroS1 status, but not with those from negative ones, indicating a good diagnostic sensitivity and specificity of the assay. The ORF2-con IHC procedure is therefore an adequate tool for the detection of BoAstV-CH13/NeuroS1 infections in cattle.

Further investigations into the single metal deposition (SMD II) technique for the detection of latent fingermarks

Single metal deposition (SMD II), a recently proposed method for the development of latent fingermarks, was investigated by systematically altering aspects of the procedure to assess their effect on the level of development and contrast achieved. Gold nanoparticle size, temperature of the deposition solution bath, and orbital shaking during detection were shown to affect the levels of development and contrast obtained. Gold nanoparticles of diameter 15–21nm were found to be most effective for satisfactory visualisation of latent fingermarks, while solutions that were applied at room temperature were found to adequately balance the ratio between the contrast of the fingermark ridge detail and the level of background staining achieved. Finally, optimum levels of development and contrast were obtained through constant agitation of both solution baths at approximately 50RPM throughout the submersion time. SMD II was also tested on a large variety of substrate types and shown to be effective on a range of porous, non-porous, and semi-porous surfaces; however, the detection quality can be significantly influenced by the substrate nature. This resulted in the production of dark grey, white, or gold coloured fingermarks on different surfaces, as well as reversed detection on certain types of plastic, similarly seen through the use of vacuum metal deposition.

Ovarian Brenner Tumors Revisited: Do We Have Space for Malignant Tumorsvand Methanol

Summary We present a case of a 75-year-old female who underwent a total abdominal hysterectomy with bilateral salpingooophorectomy for an ovarian tumor. Hematoxylin-eosin- stained slides from tumor specimen revealed 1) focci of benign Brenner tumor; 2) mucinous cysts and 3) intracystic papillary projections resembling low-grade papillary transitional cell carcinoma (Grade 1-2) with squamous differentiation and comedo-type necrosis; 4) focal areas resembling noninvasive papillary transitional cell carcinoma Grade 3. Immunohistochemical investigation with a panel of antibodies (p63, p53, Ki-67, Wilms Tumor 1 - WT1, pi 6) was initiated. Areas resembling urothelial carcinoma showed diffuse nuclear positive reaction for p63 and wild-type expression of p53. Ki-67-nuclear positivity varied from less than 5% up to 30% in areas resembling high-grade urothelial carcinoma. WT1 expression was not seen. Weak but still exceeding background staining was observed in predominantly cytoplasmic fashion with few scattered positive nuclei in transitional cell nest of the benign component. No reactivity, however, was seen within the proliferative component. The histopathological diagnosis was a borderline/atypical proliferative Brenner tumor. The patient has been regularly followed up and is at present disease-free 5 years after diagnosis. In this paper, the authors describe the morphological characteristics of Brenner tumors and address some debatable issues in the light of recent immunohistochemical and molecular studies.

Chromatographic approach for the evaluation of radical-scavenging activity using a new time-monitoring image analysis method

A thin-layer chromatographic (TLC) procedure combined with digital image processing analysis was developed for quantitative time-monitoring of the radical- scavenging activity. Reaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH•) with reference antioxidants and selected catecholamine compounds was monitored on RP-18 chromatographic plates using dot-blot technique and different concentrations of standards. Images of the chromatographic plate were recorded after background staining every 5 min for 1 h using a TLC scanner device. ImageDecipher TLC software program was used to convert image data into chromatograms, from which spot area as a function of reaction time was easily monitored for different amounts of antioxidant in all cases. For a comparative evaluation of radical-scavenging activity, all the time-acquired images were documented as a new red, green, and blue (RGB) image after conversion to different color values. Based on the chromatogram digitization results, TLC—time-monitored radical-scavenging pr...

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections.

BackgroundRust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here.ResultsTo validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues.ConclusionsThis ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

Selection of a monoclonal antibody by ELISA, immunoblotting and Quartz Crystal Microbalance technology for immunohistochemical detection of Mycoplasma mycoides subsp. mycoides.

An immunohistochemical (IHC) technique was optimised using a monoclonal antibody (MAb) to detect Mycoplasma mycoides subsp. mycoides (Mmm), the agent of Contagious Bovine Pleuropneumonia (CBPP), in sections of lung tissue. A panel of MAbs was produced and screened for Mmm speci city and for cross-reactivity against other mycoplasmas belonging and not belonging to the Mycoplasma mycoides cluster, using in parallel indirect ELISA (i-ELISA) and Immunoblotting (IB). Based on i-ELISA and IB characterization data, 1 MAb (clone 3G10E7) was selected and its highest a nity vs Mmm was con rmed by the Quartz Crystal Microbalance (QCM) technology. Afterwards, IHC analyses were conducted to compare MAb 3G10E7 vs rabbit Mmm speci c hyperimmune serum using lung tissue sections of CBPP infected and CBPP negative animals. Results suggest that screening of MAbs using in parallel ELISA, IB, and QCM technology enables to select high a nity target speci c MAbs. Immunohistochemical results demonstrated that MAb 3G10E7 improved IHC performances, showing reduced background staining and no cross-reactivity against Mycoplasma bovis, which is responsible of pneumonia in cattle.

Multispectral Fluorescence Imaging During Robot-assisted Laparoscopic Sentinel Node Biopsy: A First Step Towards a Fluorescence-based Anatomic Roadmap

BackgroundDuring (robot-assisted) sentinel node (SN) biopsy procedures, intraoperative fluorescence imaging can be used to enhance radioguided SN excision. For this combined pre- and intraoperative SN identification was realized using the hybrid SN tracer, indocyanine green-99mTc-nanocolloid. Combining this dedicated SN tracer with a lymphangiographic tracer such as fluorescein may further enhance the accuracy of SN biopsy. ObjectiveClinical evaluation of a multispectral fluorescence guided surgery approach using the dedicated SN tracer ICG-99mTc-nanocolloid, the lymphangiographic tracer fluorescein, and a commercially available fluorescence laparoscope. Design, setting, and participantsPilot study in ten patients with prostate cancer. Following ICG-99mTc-nanocolloid administration and preoperative lymphoscintigraphy and single-photon emission computed tomograpy imaging, the number and location of SNs were determined. Fluorescein was injected intraprostatically immediately after the patient was anesthetized. A multispectral fluorescence laparoscope was used intraoperatively to identify both fluorescent signatures. Surgical procedureMultispectral fluorescence imaging during robot-assisted radical prostatectomy with extended pelvic lymph node dissection and SN biopsy. Measurements(1) Number and location of preoperatively identified SNs. (2) Number and location of SNs intraoperatively identified via ICG-99mTc-nanocolloid imaging. (3) Rate of intraoperative lymphatic duct identification via fluorescein imaging. (4) Tumor status of excised (sentinel) lymph node(s). (5) Postoperative complications and follow-up. Results and limitationsNear-infrared fluorescence imaging of ICG-99mTc-nanocolloid visualized 85.3% of the SNs. In 8/10 patients, fluorescein imaging allowed bright and accurate identification of lymphatic ducts, although higher background staining and tracer washout were observed. The main limitation is the small patient population. ConclusionOur findings indicate that a lymphangiographic tracer can provide additional information during SN biopsy based on ICG-99mTc-nanocolloid. The study suggests that multispectral fluorescence image–guided surgery is clinically feasible. Patient summaryWe evaluated the concept of surgical fluorescence guidance using differently colored dyes that visualize complementary features. In the future this concept may provide better guidance towards diseased tissue while sparing healthy tissue, and could thus improve functional and oncologic outcomes.

Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues.

ABSTRACTBiomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal‐to‐noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA‐box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201–2208, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

An antibody toolkit for the study of membrane traffic in Drosophila melanogaster.

ABSTRACTThe use of Drosophila melanogaster as a model organism has been pivotal to understanding the developmental processes of metazoans. However, the use of flies for studying subcellular organization is hampered by a paucity of reliable reagents to label specific organelles. Here, we describe the generation of mouse monoclonal antibodies against a set of markers of the secretory and endocytic pathways, along with goat polyclonal antibodies against two Golgi proteins. We show that the monoclonal antibodies are highly specific and sufficiently sensitive to detect endogenous proteins in crude extracts by immunoblotting with little background staining. By immunofluorescence the major compartments of the membrane traffic system (including the endoplasmic reticulum, the Golgi, and early and late endosomes) are labeled by at least one antibody. Moreover, the antibodies can be used to label organelles in fly tissues including salivary glands and wing imaginal discs. We anticipate that these antibodies will provide a useful tool kit to facilitate the investigation of how the endomembrane system functions and varies in the diverse tissue types of metazoans.

Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

IntroductionA substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC).ResultsThe expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium.ConclusionTaken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

Novel anti-human Axl monoclonal antibodies for improved patient biomarker studies

Background: Axl receptor tyrosine kinase has been associated with poor clinical outcome in a wide variety of malignancies including breast, lung, prostate, pancreatic, brain and myeloid cancers. Axl is up-regulated during tumour epithelial-to-mesenchymal transition (EMT) and is associated with metastasis, immune evasion and drug resistance. Methods: The aim of this study was to develop improved assays for detection of Axl protein in patient samples. We developed a panel of mouse monoclonal antibodies directed against the extracellular domain of human Axl, from mice immunized with a recombinant antigen comprising the extracellular domains of human Axl fused to human IgG1 Fc domain. Anti-Axl monoclonal antibodies (MAbs) were assessed for sensitivity, specificity and utility using surface plasmon resonance (SPR) analysis, sandwich enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunohistochemistry (IHC) staining. Results: One of the anti-human Axl MAb,1H12, performed particularly well, and SPR analysis confirmed both high affinity (50 pM) and high selectivity for Axl versus other Axl-family receptors. In IHC applications on selected human carcinomas and cancer cell line pellets, MAb 1H12 showed improved sensitivity and reduced background staining compared to other members of the MAb panel. A second MAb 5F11 showed very high affinity (5.8 pM) at a non-overlapping epitope and was selected as a partner for 1H12 in sandwich ELISA, producing a highly sensitive and specific assay. Conclusion: MAbs 1H12 and 5F11 represent new anti-human Axl monoclonal antibodies for improved patient biomarker studies.

Enhanced Performance of Proliferation Assay of Bone Marrow Cells by Optimizing in vivo Incorporation of 5-Ethynyl-2′-Deoxyuridine and Cell Preparation for Flow Cytometry

ABSTRACTThe characterization of DNA synthesis and cell proliferation in vitro by 5-ethynyl-2′-deoxyuridine (EdU) incorporation is widely used, but this has not been optimized for in vivo use. In this study, the administration route, dose (0–50 mg kg−1), and incorporation duration (0–72 h) of EdU and the procedure of cell preparation for flow cytometry were optimized for in vivo proliferation assay in bone marrow cells. The percentage of EdU-positive bone marrow cells was higher following intravenous than intraperitoneal injection (p < 0.05); the percentage of positive cells increased in a dose-dependent manner from 0 to 50 mg kg−1 (p < 0.05); and the optimal dose was 25 mg kg−1. However, the percentage of positive cells was not affected by the duration of incubation in vivo from 12 to 72 h (p > 0.05). False-positive signals observed during flow cytometry were markedly reduced by subtracting the signals generated by nonviable cells stained with 7-amino-actinomycin. In addition, the percentage and mean fluorescence intensity of EdU-positive cells were increased by adding a signal enhancer to block background staining. In conclusion, the optimized procedures for in vivo incorporation of EdU and cell preparation for flow cytometry markedly improved the specificity and sensitivity of the proliferation assay of bone marrow cells.

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.