scholarly journals Quenching autofluorescence in tissue immunofluorescence

2017 ◽  
Vol 2 ◽  
pp. 79 ◽  
Author(s):  
Jian Yang ◽  
Fengtang Yang ◽  
Lia S. Campos ◽  
William Mansfield ◽  
Helen Skelton ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Poor Quality ◽  
Frozen Sections ◽  
Combined Treatments ◽  
Tissue Sections ◽  
Sudan Black B ◽  
Specific Proteins ◽  
Teratoma Formation ◽  
Texas Red ◽  
Embryonic Organ

Background: Immunofluorescence (IF) is one of the most important techniques where fluorochromes conjugated to antibodies are used to detect specific proteins or antigens. In tissue sections, autofluorescence (AF) can lead to poor quality images that impair assessment. The placenta is a pivotal extra-embryonic organ in embryo development, where trophoblasts make up a large proportion of the cells. Teratoma formation is one of the critical assays for pluripotent stem cells. Methods: We tested whether ultraviolet (UV), ammonia (NH3), copper (II) sulfate (CuSO4), Trypan Blue (TB), Sudan Black B (SB), TrueBlack™ Lipofusin Autofluorescence Quencher (TLAQ) and combinations of these treatments could reduce AF in paraffin and frozen sections of placenta and teratoma in FITC, Texas Red and Cy5.5 channels. Results: We found that UV, NH3, TB and CuSO4 quenched AF to some extent in different tissue and filters, but increased AF in Texas Red or Cy5.5 channels in some cases. SB and TLQA exhibited the most consistent effects on decreasing AF, though TLQA reduced the overall IF signal in placenta sections. Not all combined treatments further reduced AF in both placenta and teratoma sections. Conclusions: SB and TLAQ can effectively quench AF in placenta and teratoma IF.

RESEARCH ON VALUE OF INTRAOPERATIVE FROZEN SECTION EXAMINATION OF THE COMMON TUMORS AT HUE UNIVERSITY HOSPITAL

2011 ◽  
pp. 67-73
Author(s):  
Cong Thuan Dang ◽  
Thi Thu Thao Le
Keyword(s):  
Sensitivity And Specificity ◽  
Frozen Section ◽  
Analysis Data ◽  
Poor Quality ◽  
University Hospital ◽  
Frozen Sections ◽  
Intraoperative Frozen Section ◽  
Frozen Section Examination ◽  
Incorrect Diagnosis ◽  
The Common

Background: To evaluate the accuracy and the pitfalls of frozen section examination in diagnosis the common tumors at Hue University Hospital. Materials and method: A retrospective analysis data of 99 consecutive patients from 2007 to 2009 were evaluated and analyzed the major pitfalls. In our 99 patients, 100% cases we compared histological diagnosis on frozen sections with those on paraffin sections. Results: The majority of frozen section examinations were the thyroid lesions 37.4%, breast lesions 25.2%, lymph nodes 16.1%, ovary 9.1% and less common in other diseases (12.1%). The accuracy, sensitivity and specificity of the intraoperative frozen section examination were 93.9%, 89.1% and 98.1% respectively. The main factors causing incorrect diagnosis in frozen section are: Misinterpretation, poor quality of frozen sections, improper sampling in sectioning and difficult to result interpretation. Conclusion: The frozen section analysis of suspect lesions displays good sensitivity and specificity characteristics.

scholarly journals Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 876
Author(s):  
Raquel Bernad ◽  
Cian J. Lynch ◽  
Rocio G. Urdinguio ◽  
Camille Stephan-Otto Attolini ◽  
Mario F. Fraga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Normal Karyotype ◽  
Cell Types ◽  
Dna Demethylation ◽  
Starting State ◽  
Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

scholarly journals PSII-35 Melatonin supplementation and restricted nutrition do not affect chorionic somatomammotropin (CSH) concentration in ovine placenta from mid- to late- gestation

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 244-244
Author(s):  
Hanaa Mansour ◽  
Caleb O Lemley ◽  
Russell Anthony ◽  
Kendall C Swanson ◽  
Anna T Grazul-Bilska ◽  
...  
Keyword(s):  
Histological Analysis ◽  
Late Gestation ◽  
Fetal Membrane ◽  
Nutrient Restriction ◽  
Binucleate Cells ◽  
Tissue Sections ◽  
Vasoactive Factors ◽  
Ovine Placenta ◽  
Chorionic Somatomammotropin ◽  
Texas Red

Abstract Melatonin plays a role as a vasodilator. Vasoactive and angiogenic factors are expressed by placental binucleate cells (BNC) and produce chorionic somatomammotropin (CSH), known to impact fetal and placental growth. We hypothesized that melatonin supplementation and restricted nutrition from mid- to late-gestation would alter CSH concentration and some characteristics of BNC in placenta. At day 50 of gestation, ewes carrying singletons were randomly assigned to a 2 × 2 factorial design and were fed either an adequate (ADQ; 100% NRC; n = 15) or restricted (RES; 60% NRC; n = 15) diet supplemented with 0 (CON, n = 14) or 5 mg of melatonin (MEL; n = 16). Placentomes were collected on day 130 of gestation and preserved in formalin for histological analysis. Cotyledon (COT) were snap frozen for western immunoblotting analyses. Tissue sections were stained using biotinylated Dolichos Biflurus (DBA; a marker of fetal membrane) lectin and fluorescein labeled Texas red-avidin and fluorescein labeled Griffonia Simplifolica (BS) lectin (a marker of BNC). The number, area, and diameter of BNC in COT were determined by image analysis. For immunoblotting, protein was extracted from COT in SDS phosphate buffer, loaded equally, and separated on 12.5% polyacrylamide gels. Protein was transferred to PVDF membranes and incubated with rabbit anti-CSH. Bands were visualized and imaged. Data were analyzed using Proc Mixed procedure of SAS. Melatonin supplementation and restricted nutrition did not affect BNC number, area, or diameter, or CSH protein expression. While we reject our hypothesis that melatonin supplementation and nutrient restriction would alter the CSH concentration and BNC characteristics in COT, we continue to evaluate if the BNC produce angiogenic or vasoactive factors that may influence placental and mammary gland functions in sheep.

scholarly journals Generation of Engraftable Hematopoietic Stem Cells From Induced Pluripotent Stem Cells by Way of Teratoma Formation

2013 ◽  
Vol 21 (7) ◽  
pp. 1424-1431 ◽  
Author(s):  
Nao Suzuki ◽  
Satoshi Yamazaki ◽  
Tomoyuki Yamaguchi ◽  
Motohito Okabe ◽  
Hideki Masaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Stem ◽  
Induced Pluripotent ◽  
Teratoma Formation

Sudan Black B Reduces Autofluorescence in Murine Renal Tissue

2011 ◽  
Vol 135 (10) ◽  
pp. 1335-1342 ◽  
Author(s):  
Yan Sun ◽  
Hong Yu ◽  
Dong Zheng ◽  
Qi Cao ◽  
Ya Wang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Kidney Tissue ◽  
Fluorescein Isothiocyanate ◽  
Renal Tissue ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Frozen Sections ◽  
Paraffin Sections ◽  
Biopsy Material ◽  
Sudan Black B

Context.—Renal tissue emits intense autofluorescence, making it difficult to differentiate specific immunofluorescence signals and thus limiting its application to clinical biopsy material. Objective.—To identify and minimize autofluorescence of renal tissue and demonstrate a simple, efficient method to reduce autofluorescence using Sudan black B. Design.—In this study, the sources and features of autofluorescence emitted from kidney tissue were examined. Broad autofluorescence was visualized in both frozen and paraffin kidney sections of normal mice and mice with Adriamycin-induced nephropathy using confocal laser scanning microscopy. Autofluorescence appeared in commonly used 4′,6-diamidino-2-phenylindole, fluorescein isothiocyanate, and Texas Red channels but not in far-red channel, and emitted extensively from red cells, injured tubulointersitial cells, and protein casts in diseased kidney. To eliminate autofluorescence, Sudan black B was used on formaldehyde-fixed paraffin sections and frozen sections of mouse kidney. The effects of Sudan black B in various concentrations were tested on kidney tissue. Results.—The 0.1% Sudan black B effectively blocked autofluorescence from both paraffin and frozen sections without adversely affecting specific fluorescence signals. Interestingly, the solvent for Sudan black B, 70% ethanol, was also shown to reduce autofluorescence on frozen sections, but not on paraffin sections. Conclusions.—This study demonstrates a simple, efficient, and cost-effective method to reduce autofluorescence using Sudan black B, and also provides a comprehensive approach to identify and minimize autofluorescence of renal tissue.

scholarly journals Tumorigenicity of pluripotent stem cells: biological insights from molecular imaging

2010 ◽  
Vol 7 (suppl_6) ◽  
Author(s):  
Nigel G. Kooreman ◽  
Joseph C. Wu
Keyword(s):  
Stem Cells ◽  
Molecular Imaging ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cell Replacement Therapy ◽  
Cell Replacement ◽  
Induced Pluripotent ◽  
Teratoma Formation

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the ability (i) to duplicate indefinitely while maintaining pluripotency and (ii) to differentiate into cell types of all three embryonic germ layers. These two properties of ESCs and iPSCs make them potentially suitable for tissue engineering and cell replacement therapy for many different diseases, including Parkinson's disease, diabetes and heart disease. However, one critical obstacle in the clinical application of ESCs or iPSCs is the risk of teratoma formation. The emerging field of molecular imaging is allowing researchers to track transplanted ESCs or iPSCs in vivo , enabling early detection of teratomas.

scholarly journals Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity

2018 ◽  
Author(s):  
Nelly Olova ◽  
Daniel J Simpson ◽  
Riccardo Marioni ◽  
Tamir Chandra
Keyword(s):  
Pluripotent Stem Cells ◽  
Tissue Replacement ◽  
Age Related ◽  
Epigenetic Age ◽  
Somatic Gene ◽  
Induced Pluripotent ◽  
Teratoma Formation ◽  
Human Ipsc ◽  
Risk Of Cancer

SummaryInduced pluripotent stem cells (IPSCs), with their unlimited regenerative capacity, carry the promise for tissue replacement to counter age-related decline. However, attempts to realise in vivo iPSC have invariably resulted in the formation of teratomas. Partial reprogramming in prematurely aged mice has shown promising results in alleviating age-related symptoms without teratoma formation. Does partial reprogramming lead to rejuvenation (i.e. “younger” cells), rather than dedifferentiation, which bears the risk of cancer? Here we analyse the dynamics of cellular age during human iPSC reprogramming and find that partial reprogramming leads to a reduction in the epigenetic age of cells. We also find that the loss of somatic gene expression and epigenetic age follow different kinetics, suggesting that they can be uncoupled and there could be a safe window where rejuvenation can be achieved with a minimised risk of cancer.

scholarly journals Magnetically Promoted Rapid Immunofluorescence Staining for Frozen Tissue Sections

2019 ◽  
Vol 67 (8) ◽  
pp. 575-587 ◽  
Author(s):  
Tatsuya Onishi ◽  
Sachiko Matsuda ◽  
Yuki Nakamura ◽  
Junko Kuramoto ◽  
Akinori Tsuruma ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Growth Factor Receptor ◽  
Metastatic Breast ◽  
Detection Methods ◽  
Frozen Sections ◽  
Tissue Sections ◽  
Tissue Arrays ◽  
Abnormal Cells ◽  
Epidermal Growth ◽  
Hematoxylin And Eosin

Current immunohistochemistry methods for diagnosing abnormal cells, such as cancer cells, require multiple steps and can be relatively slow compared with intraoperative frozen hematoxylin and eosin staining, and are therefore rarely used for intraoperative examination. Thus, there is a need for novel rapid detection methods. We previously demonstrated that functionalized fluorescent ferrite beads (FF beads) magnetically promoted rapid immunoreactions. The aim of this study was to improve the magnetically promoted rapid immunoreaction method using antibody-coated FF beads and a magnet subjected to a magnetic field. Using frozen sections of xenograft samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-coated FF beads, we reduced the magnetically promoted immunohistochemistry procedure to a 1-min reaction and 1-min wash. We also determined the optimum magnetic force for the antibody reaction (from 7.79 × 10−15 N to 3.35 × 10−15 N) and washing (4.78 × 10−16 N), which are important steps in this technique. Furthermore, we stained paraffin-embedded tissue arrays and frozen sections of metastatic breast cancer lymph nodes with anti-pan-cytokeratin antibody-coated FF beads to validate the utility of this system in clinical specimens. Under optimal conditions, this ultra-rapid immunostaining method may provide an ancillary method for pathological diagnosis during surgery. (J Histochem Cytochem 58:XXX–XXX, 2010)

Quantitative Comparative Immunohistology

1969 ◽  
Vol 15 (6) ◽  
pp. 505-508 ◽  
Author(s):  
Martin Semar ◽  
Gerhard Treser ◽  
Kurt Lange
Keyword(s):  
Biopsy Specimen ◽  
Quantitative Assay ◽  
Tissue Sections ◽  
Specific Proteins ◽  
Resultant Solution

Abstract A quantitative assay for the determination of fluorescence of tissue sections stained with fluorescein-labeled antibodies using a fluorometer is described. Sequential microtome sections were stained with labeled antiserums, washed repeatedly with buffered saline, and digested with 20% (w/v) NaOH at 60° for 12 hr. The fluorescence of the resultant solution is then read in a fluorometer. There is similarity between sequential sections of the same biopsy specimen. Specific blocking procedures reduce the readings of the fluorescence markedly. By comparison with values obtained simultaneously from standard curves of the labeled antiserums used, the amount of bound antibody or specific proteins can be determined quantitatively.

Sign in / Sign up

Export Citation Format

Share Document