scholarly journals Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)

2001 ◽  
Vol 49 (12) ◽  
pp. 1565-1571 ◽  
Author(s):  
Werner Baschong ◽  
Rosmarie Suetterlin ◽  
R. Hubert Laeng
Keyword(s):  
Bone Marrow ◽  
Sodium Borohydride ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Frozen Sections ◽  
Paraffin Sections ◽  
Fixed Tissue ◽  
Tissue Processing ◽  
Sudan Black B

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.

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.

A New Approach to Phenotyping Disseminated Tumor Cells: Methodological advances and Clinical Implications

2000 ◽  
Vol 15 (1) ◽  
pp. 100-104 ◽  
Author(s):  
F. Noack ◽  
M. Schmitt ◽  
J. Bauer ◽  
D. Helmecke ◽  
W. Krüger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Laser Scanning ◽  
Simultaneous Detection ◽  
Disseminated Tumor Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Prognostic Impact ◽  
Primary Therapy ◽  
Double Immunofluorescence

At the time of primary therapy (surgery, systemic chemotherapy and/or radiation), disseminated tumor cells in the bone marrow can be found in almost one-third of patients with cancer of the breast, ovary, esophagus, stomach, colon, and other solid tumors. Whereas the prognostic impact of the mere presence of these cells is still a matter of debate, it has been shown that expression of tumor-associated antigens in disseminated tumor cells is linked to more aggressive disease. Therefore, further characterization of disseminated tumor cells at the protein and gene level has become increasingly important. To date, the most common detection method for disseminated tumor cells in the bone marrow is an immunocytochemical approach using cytokeratin-directed antibodies for detection of epithelial cells and the APAAP system for their visualization. We have established a new double immunofluorescence technique enabling simultaneous detection, phenotyping, and antigen quantification of disseminated tumor cells. Mononuclear cells from bone marrow are enriched by Ficoll gradient centrifugation and cytospins are prepared. Double immunofluorescence is performed using antibodies against cytokeratins 8/18/19 (mAb A45B/B3) and the uPA receptor CD87 (pAb HU277). CD87 expression is recorded by confocal laser scanning microscopy (CLSM) using fluorescence labeled latex beads as the reference; staining intensities of all the scans are then summed and quantified (extended focus). This protocol, originally designed for disseminated tumor cells in bone marrow, can also be applied to disseminated tumor cells in blood, to leukapheresis cells or to cells present in malignant ascites or other malignant effusions. The tumor cells detected may be used for gene and mRNA analyses. Furthermore, disseminated tumor cells also represent interesting targets for clinical studies on patient prognosis or prediction of therapy response as well as for specific tumor-biological therapies.

scholarly journals CXCL12-Abundant Reticular (CAR) Cells Direct Megakaryocyte Protrusions Across the Bone Marrow Sinusoid Wall

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 722
Author(s):  
Nicole Wagner ◽  
Kristina Mott ◽  
Berin Upcin ◽  
David Stegner ◽  
Harald Schulze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Circulation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Tissue Sections ◽  
Complex Process ◽  
Block Face ◽  
Platelet Release ◽  
Cell Retraction

Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.

scholarly journals Elimination of Autofluorescence in Archival Formaldehyde-Fixed, Paraffin-Embedded Bone Marrow Biopsies

2018 ◽  
Vol 143 (3) ◽  
pp. 362-369 ◽  
Author(s):  
Wenxia Su ◽  
Lin Yang ◽  
Xueping Luo ◽  
Meng Chen ◽  
Jinqin Liu
Keyword(s):  
Bone Marrow ◽  
Efficient Method ◽  
Laser Scanning ◽  
Primary Myelofibrosis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Immunofluorescence Analysis ◽  
Bone Marrow Biopsies ◽  
Hematologic Disorders ◽  
Paraffin Tissue

Context.— High levels of autofluorescence in bone marrow tissue constitute a major obstacle to immunofluorescence analysis of bone marrow biopsies. Objective.— To present a simple, efficient method to eliminate autofluorescence in bone marrow biopsies. Design.— Autofluorescence of paraffin bone marrow tissues was examined in different hematologic disorders with confocal laser scanning microscopy. Strong autofluorescence was observed in primary myelofibrosis and acute leukemia with reticulin myelofibrosis in 488-nm and 561-nm channels. To eliminate autofluorescence, AutoFluo Quencher was used on bone marrow sections with different incubation times. The effects of AutoFluo Quencher on immunofluorescence analysis of bone marrow biopsies was tested using antibodies tagged with different fluorophores. Results.— AutoFluo Quencher thoroughly eliminated the strong autofluorescence of bone marrow but did not decrease the intensity of fluorophores, leaving the specific signals of target proteins clearly visible. Conclusions.— This study presents a simple, efficient method to eliminate autofluorescence in bone marrow paraffin tissue, and it opens the way to better results in the immunofluorescence analysis of bone marrow biopsies.

scholarly journals Characterization and Quenching of Autofluorescence in Piglet Testis Tissue and Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Yanfei Yang ◽  
Ali Honaramooz
Keyword(s):  
Laser Scanning ◽  
Intrinsic Fluorescence ◽  
Laser Scanning Microscopy ◽  
Epifluorescence Microscopy ◽  
Fluorescence Labeling ◽  
Confocal Laser ◽  
Target Cells ◽  
Tissue Sections ◽  
Sudan Black B ◽  
Testis Tissue

Significant intrinsic fluorescence in tissues and in disassociated cells can interfere with fluorescence identification of target cells. The objectives of the present study were (1) to examine an intrinsic fluorescence we observed in both the piglet testis tissue and cells and (2) to test an effective method to block the autofluorescence. We observed that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry. The emission wavelength of the autofluorescent substance ranged from 425 to 700 nm, a range sufficiently broad that could potentially interfere with fluorescence techniques. When we treated the samples with Sudan Black B for different incubation times, the intrinsic fluorescence was completely masked after treatment for 10–15 min of the testis tissue sections or for 8 min of the testis cells, without compromising specific fluorescence labeling of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin or lipofuscin-like pigments within Leydig cell granules were mainly responsible for the observed intrinsic fluorescence in piglet testes. The method described in the present study can facilitate the identification and characterization of piglet gonocytes using fluorescence microscopy.

scholarly journals Ex Vivo and in Vivo Study of Sucrosomial® Iron Intestinal Absorption and Bioavailability

2018 ◽  
Vol 19 (9) ◽  
pp. 2722 ◽  
Author(s):  
Angela Fabiano ◽  
Elisa Brilli ◽  
Letizia Mattii ◽  
Lara Testai ◽  
Stefania Moscato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Laser Scanning ◽  
Time Course ◽  
Ex Vivo ◽  
Iron Bioavailability ◽  
Laser Scanning Microscopy ◽  
Raw Material ◽  
Confocal Laser ◽  
Ussing Chambers

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.

scholarly journals V617FJAK2-Positive Endothelial Cells Are Present in Bone Marrow Neovessels of Patients with Myelofibrosis and Could Derive from the Transdifferentiation of Mutated Hematopoietic Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2833-2833
Author(s):  
Laura Villani ◽  
Rita Campanelli ◽  
Gabriella Gaudioso ◽  
Valentina Poletto ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Laser Scanning ◽  
Laser Microdissection ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Allelic Burden

Abstract Background. Extensive neoangiogenesis is a distinctive feature of both the spleen and the bone marrow (BM) of patients with myelofibrosis (MF). We and others have previously reported that mature endothelial cells obtained from microvessels of the spleen, either by laser microdissection or by cell sorting, harbor the V617FJAK2 mutation, whereas nothing is known about the existence of such mutated endothelial cells in the BM. The origin of the mutated endothelium has not been clarified up to now, although it has been proposed that it could derive from the transdifferentiation of a mutated CD34+ hematopoietic progenitor cells into an endothelial cell, an event similar to what already observed in solid tumors. Aim. To assess the presence of the V617FJAK2 mutation in mature endothelial cells from the BM of patients with MF and to evaluate the presence of transitional cells expressing both myeloid and endothelial cell markers in the BM of MF patients. Patients and Methods. Paraffin-embedded BM sections from 5 patients with MF, harboring a V617FJAK2 mutation in their granulocytes, were used both for isolating mature endothelial cells by laser microdissection and for immunostaining with antibodies directed against CD34, CD33, and CD144 (VE-cadherin) antigens. Sections from patients with lymphomas who underwent a BM biopsy for staging of the disease were used as controls (n=2). The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGE J software. DNA was extracted from 50 microdissected endothelial cells by a commercial kit and the presence of V617FJAK2 mutation assessed by an allele-specific real-time quantitative PCR. Results. The V617FJAK2 mutation was detected in at least 1 sample of endothelial cells of 5/5 patients, indicating that a mutated endothelium, in addition to the spleen, is also detectable in the BM of patients with MF. The allelic burden ranged from 51.4% to 84.1% of mutated alleles, and was similar to the allelic burden of hematopoietic cells in 1 case, whereas it was lower in 4. Staining of BM sections from 4 patients with antibodies specific for hematopoiesis (CD33) and endothelium (VE-cadherin), as well as common to the 2 lineages (CD34), while confirming the presence of increased neoangiogenetic processes in samples from MF patients, allowed the detection of cells, usually located close to neovessels, co-expressing both hematopoietic (CD33, CD34) and endothelial (VE-cadherin, CD34) markers. These "triple positive" cells were found in all the slides of all patients whereas they were never observed in the BM sections of controls. Conclusions. V617FJAK2 positive endothelial cells can be detected in the BM of patients with MF, similarly to what observed in the spleen. These mutated endothelial cells could derive from the transdifferentiation of hematopoietic mutated cells as suggested by the detection of cells co-expressing both myeloid and endothelial markers. These observations support the hypothesis that the endothelial lineage is involved in the process of malignant transformation of MF, providing new perspectives in our understanding of the phenotype, evolution, and therapy of the disease. Disclosures No relevant conflicts of interest to declare.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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