staining quantification
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We describe a detailed embedding procedure for large bone specimens in methyl methacrylate and a new staining method by which thin sections (appr 100 um) of undecalcified bones with synthetic implants can be coloured. Different staining effects were obtained which greatly facilitated the evaluation of sections with bone, new forming bone and especially remnants of synthetic implants. The identification and quantification of the latter is difficult in common staining techniques. A detailed embedding - staining - mounting procedure is proposed.
Quantification of microvessels in tumors is mostly based on counts of vessel profiles in tumor hot spots. Drawbacks of this method include low reproducibility and large interobserver variance, mainly as a result of individual differences in sampling of image fields for analysis. Our aim was to test an unbiased method for quantifying microvessels in healthy and tumorous lymph nodes of dogs. The endothelium of blood vessels was detected in paraffin sections by a combination of immunohistochemistry (von Willebrand factor) and lectin histochemistry (wheat germ agglutinin) in comparison with detection of basal laminae by laminin immunohistochemistry or silver impregnation. Systematic uniform random sampling of 50 image fields was performed during photo-documentation. An unbiased counting frame (area 113,600 microm(2)) was applied to each micrograph. The total area sampled from each node was 5.68 mm(2). Vessel profiles were counted according to stereological counting rules. Inter- and intraobserver variabilities were tested. The application of systematic uniform random sampling was compared with the counting of vessel profiles in hot spots. The unbiased estimate of the number of vessel profiles per unit area ranged from 100.5 +/- 44.0/mm(2) to 442.6 +/- 102.5/mm(2) in contrast to 264 +/- 72.2/mm(2) to 771.0 +/- 108.2/mm(2) in hot spots. The advantage of using systematic uniform random sampling is its reproducibility, with reasonable interobserver and low intraobserver variance. This method also allows for the possibility of using archival material, because staining quality is not limiting as it is for image analysis, and artifacts can easily be excluded. However, this method is comparatively time-consuming. (c) 2008 Wiley-Liss, Inc.
- MeSH
- barvení a značení MeSH
- biometrie metody MeSH
- cévní endotel MeSH
- cévy anatomie a histologie patologie MeSH
- imunohistochemie metody MeSH
- lymfatické uzliny anatomie a histologie patologie MeSH
- nádory patologie MeSH
- patologie metody MeSH
- psi MeSH
- reprodukovatelnost výsledků MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- hodnotící studie MeSH
Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.
- MeSH
- barvení a značení metody MeSH
- buněčná adheze MeSH
- buněčné linie MeSH
- buněčný cyklus MeSH
- cytofotometrie metody MeSH
- diploidie * MeSH
- DNA analýza chemie MeSH
- dodecylsíran sodný chemie MeSH
- fluorescenční barviva chemie MeSH
- HeLa buňky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- počet buněk přístrojové vybavení metody MeSH
- replikace DNA * MeSH
- reprodukovatelnost výsledků MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
U pacientů s vlasatobuněčnou leukémií, léčených 2-chlorodeoxyadenosinem nebo 2-deoxycoformicinem, je dosahováno velmi vysokého počtu terapeutických odpovědí. Při dosažení kompletní remise - minimální reziduálni nemoci nelze prokázat aktivitu onemocnění, splenomegalii a lymfadenopatii; dochází k normalizaci koncentrace hemoglobinu, počtu granulocytů a krevních destiček. Současně nelze standardním barvením prokázat leukemické buňky v periferní krvi a v kostní dřeni. K jejich průkazu lze použít imunohistochemického vyšetření pomocí protilátky DBA.44 na intenzitu imunohistochemické reakce a zároveň morfologii vlasatých buněk. Při kvantifikaci leukemických buněk je používána počítačová analýza obrazu LUCIA-M.
Hairy cell leukemia patients treated with 2-chlorodeoxyadenosine or 2-deoxycoformicin achieve a veiy high number of therapeutic responses. After complete remission, i.e. minimal residual disease, we cannot demonstrate the disease activity, splenomegaly, or lymphadenopathy; moreover, there comes to normalization of hemoglobin concentration, leukocyte count, and platelet count. No leukemic cells in peripheral blood or bone marrow smears can be seen with the use of staining techniques. They can be demonstrated immunohistochemically in decalcified trephine bone marrow biopsies with the use of DBA.44 antibody together with their morphologic features. For quantification of leukemic cells we use LUCIA-M image analysis.
Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO(®)-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO(®)-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm.
- MeSH
- barvení a značení metody MeSH
- biofilmy růst a vývoj MeSH
- Candida fyziologie MeSH
- Escherichia coli fyziologie MeSH
- karbocyaniny metabolismus MeSH
- mikrobiální viabilita * MeSH
- mikrobiologické techniky metody MeSH
- průtoková cytometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
For liver fibrosis assessment, the liver biopsy is usually stained with Masson's trichrome (MT) or picrosirius red (PSR) to quantify liver connective tissue (LCT) for fibrosis scoring. However, several concerns of such semiquantitative assessments have been raised, and when searching for data on the amount of LCT in healthy rats, the results vastly differ. Regarding the ongoing reproducibility crisis in science, it is necessary to inspect the results and methods, and to design an unbiased and reproducible method of LCT assessment. We searched the Medline database using search terms related to liver fibrosis, LCT and collagen, rat strains, and staining methods. Our search identified 74 eligible rat groups in 57 studies. We found up to 170-fold differences in the amount of LCT among healthy Wistar and Sprague-Dawley rats, with significant differences even within individual studies. Biased sampling and quantification probably caused the observed differences. In addition, we also found incorrect handling of liver fibrosis scoring. Assessment of LCT using stereological sampling methods (such as systematic uniform sampling) would provide us with unbiased data. Such data could eventually be used not only for the objective assessment of liver fibrosis but also for validation of noninvasive methods of the assessment of early stages of liver fibrosis.
- Publikační typ
- časopisecké články MeSH
The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators—palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)—in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM’s analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography–tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3–5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.
- Publikační typ
- časopisecké články MeSH
The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators-palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)-in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM's analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3-5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.
- Publikační typ
- časopisecké články MeSH
