scholarly journals Technique and Feasibility of a Dual Staining Method for Estrogen Receptors and AgNORs

2000 ◽  
Vol 20 (4) ◽  
pp. 151-154 ◽  
Author(s):  
Lukas Günther ◽  
Peter Hufnagl

A new staining method for dual demonstration of Estrogen receptors (ER) and argyrophilc Nucleolus‐Organizer Regions (AgNORs) was developed. To rule out possible reciprocal effects, serial slides of 10 invasive ductale breast cancers were stained with either the single staining method or the simultaneous ER/AgNOR‐staining method and investigated comparatively. By measuring the slides with the image analysis system AMBA, reciprocal effects could be excluded. It was proven that dual staining of both markers results in a reproducible and specific staining result. We concluded that it is justified to measure AgNORs in immunohistochemically stained cells.

1990 ◽  
Vol 38 (11) ◽  
pp. 1591-1597 ◽  
Author(s):  
J Foucrier ◽  
J P Rigaut ◽  
D Pechinot

We describe a new staining technique (H-Ag-S) which allows observation and counting of active nucleolus organizer regions (NORs) and evaluation of the amount of DNA in the same cell nucleus. The procedure consists of combining a modified AgNOR staining method with the Feulgen reaction. A sequential procedure is proposed, based on the determination of optimal staining conditions. The technique, which was designed to allow studies of correlations between the transcriptional activity of rDNA genes and the cell ploidy, was primarily developed for rat liver smears. It should be applicable to most biological preparations, but the optimal conditions might be variable.


1996 ◽  
Vol 44 (9) ◽  
pp. 1043-1050 ◽  
Author(s):  
D E Mosedale ◽  
J C Metcalfe ◽  
D J Grainger

There is a growing trend towards the objective quantification of immunohistochemical staining. However, quantification has not been used previously to optimize the original published immunohistochemical methods. We present a quantitative method for analyzing immunofluorescence staining employing the Applied Imaging MAGISCAN image analysis system, which has then been used to optimize major aspects of the standard immunofluorescent staining protocols. The optimization process resulted in a method that increased specific staining up to fivefold over typical published protocols, with no increase in nonspecific staining. The method is extremely reproducible. For slides stained by a single experimenter in one batch on one day, the coefficient of variation between replicate means is 1.2%. The image analysis protocol gave a linear response with increasing antigen concentration, as determined by using purified antigen dried onto slides. The revisions to the standard protocol presented here can also be applied to nonquantitative staining. It will help users of immunofluorescence to maximize their staining and may enable the detection of previously undetected antigens.


1985 ◽  
Vol 33 (1) ◽  
pp. 11-20 ◽  
Author(s):  
D S Gross ◽  
J M Rothfeld

A limiting factor in the use of immunocytochemistry in experimental endocrine studies has been the lack of a suitable procedure for quantification of immunoreactive hormones. The objective of the present study was the development of an automated, computerized image analysis system adapted to the quantitative analysis of light microscopic immunocytochemical reaction product. Reaction conditions that result in optimum, standardized, and quantitatively linear development of reaction deposit are described for H2O2 and diaminobenzidine concentrations, antiserum dilutions, and substrate incubation times. In addition, evaluation techniques, including the use of a standard control section to monitor variance and incorporate it into the statistical analysis of the results are documented. For each of the reaction variables, the immunostaining was linear over the range of specific staining. When the optimum conditions were exceeded, marked over-estimations of hormone levels occurred due to the detection of nonspecific background features reaching the detection threshold. Application of this quantitative immunocytochemical (QICC) method to the analysis of variations in hypothalamic and pituitary hormone levels was validated by comparing values obtained with QICC to those with radioimmunoassay (RIA). The relative changes in both hypothalamic gonadotropin-releasing hormone and pituitary luteinizing hormone induced by manipulation of gonadal steroid levels, as measured by RIA and QICC, were highly correlated. Two-way analysis of variance revealed that the two techniques were not significantly different in their detection of changes in either hormone. Thus, under optimally defined conditions, quantitative immunocytochemistry using computerized image analysis has been validated for the accurate measurement of pituitary and brain hormones in precise regions.


Author(s):  
A. V. Crewe ◽  
M. Ohtsuki

We have assembled an image processing system for use with our high resolution STEM for the particular purpose of working with low dose images of biological specimens. The system is quite flexible, however, and can be used for a wide variety of images.The original images are stored on magnetic tape at the microscope using the digitized signals from the detectors. For low dose imaging, these are “first scan” exposures using an automatic montage system. One Nova minicomputer and one tape drive are dedicated to this task.The principal component of the image analysis system is a Lexidata 3400 frame store memory. This memory is arranged in a 640 x 512 x 16 bit configuration. Images are displayed simultaneously on two high resolution monitors, one color and one black and white. Interaction with the memory is obtained using a Nova 4 (32K) computer and a trackball and switch unit provided by Lexidata.The language used is BASIC and uses a variety of assembly language Calls, some provided by Lexidata, but the majority written by students (D. Kopf and N. Townes).


Author(s):  
D.S. DeMiglio

Much progress has been made in recent years towards the development of closed-loop foundry sand reclamation systems. However, virtually all work to date has determined the effectiveness of these systems to remove surface clay and metal oxide scales by a qualitative inspection of a representative sampling of sand particles. In this investigation, particles from a series of foundry sands were sized and chemically classified by a Lemont image analysis system (which was interfaced with an SEM and an X-ray energy dispersive spectrometer) in order to statistically document the effectiveness of a reclamation system developed by The Pangborn Company - a subsidiary of SOHIO.The following samples were submitted: unreclaimed sand; calcined sand; calcined & mechanically scrubbed sand and unused sand. Prior to analysis, each sample was sprinkled onto a carbon mount and coated with an evaporated film of carbon. A backscattered electron photomicrograph of a field of scale-covered particles is shown in Figure 1. Due to a large atomic number difference between sand particles and the carbon mount, the backscattered electron signal was used for image analysis since it had a uniform contrast over the shape of each particle.


2018 ◽  
Author(s):  
F.B. Musaev ◽  
N.S. Priyatkin ◽  
M.V. Arkhipov ◽  
P.A. Shchukina ◽  
A.F. Bukharov ◽  
...  

Приведено описание разработанной авторами методики цифровой компьютерной морфометрии семян овощных культур на основе системы анализа изображений, состоящей из планшетного сканера и программного обеспечения для автоматических измерений. В основу метода положено представление о разнокачественности семян, обусловленной генетической неоднородностью самих семенных растений, используемых в промышленном семеноводстве. Физические свойства семян (их форма и линейные размеры) – основные параметры при определении их качества. Цифровые изображения семян получены при помощи планшетного сканера HP Sсanjet 200 на базе Агрофизического НИИ с использованием серийного программного обеспечения «Argus-BIO», производства ООО «АргусСофт» (г. Санкт-Петербург). Метод состоит из подбора контрастной подложки (фона) для сканирования семян с минимальными теневыми эффектами, калибровку программного обеспечения для привязки к истинным размерным величинам, подбор параметров измерений и автоматическое распознавание цифровых сканированных изображений семян. Представлены экспериментальные данные по морфометрии экологически разнокачественных семян фасоли овощной, матрикально разнокачественных семян укропа, пастернака и лука Кристофа. Семена укропа и пастернака, собранные из разных порядков ветвления семенного растения, значительно различались по величине линейных параметров. Наиболее показательный линейный параметр семян – площадь проекции. Предложенная авторами методика цифровой морфометрии, уже использована на практике и в перспективе может быть задействована в исследованиях экологической и матрикальной разнокачественности семян овощных культур. Так, она прошла апробацию на разнокачественных семенах пяти сортов фасоли овощной (Настена, Магура, Миробела, Морена, Бажена) полученных в пяти контрастных эколого-географических условиях среды (Москва, Белгород, Ставрополь, Омск, Горки) в 2011–2012 годах. В дальнейшем методика может быть использована для улучшения качества цифровых изображений семян, изучения разнокачественности семян в том числе и для совершенствования контроля за селекционным процессом. Кроме того, она применима для изучения взаимосвязи совокупности морфометрических характеристик семян и их посевных качеств.The description of the method of digital computer morphometry of vegetable seeds developed by the authors on the basis of the image analysis system consisting of a flatbed scanner and software for automatic measurements is given. The method is based on the idea of seed quality, due to the genetic heterogeneity of the seed plants used in industrial seed production. Physical properties of seeds (their shape and linear dimensions) are the main parameters in determining their quality. Digital image of the seed obtained using the flatbed scanner, HP Sсanjet 200 on the basis of the Agrophysical research Institute with serial software “Argus-BIO”, produced by LLC “Argussoft” (Saint-Petersburg). The method consists of selection of a contrast substrate (background) for scanning seeds with minimal shadow effects, calibration of software for binding to true size values, selection of measurement parameters and automatic recognition of digital scanned images of seeds. Experimental data on the morphometry of ecologically different-quality seeds of vegetable beans, matrix seeds of dill, Pasternak and Christoph onion are presented. Seeds of dill and parsnip, collected from different orders of branching of the seed plant, significantly differed in size of linear parameters. The most revealing linear parameter seed – area projection. The method of digital morphometry proposed by the authors has already been used in practice and in the future can be used in studies of ecological and matrix heterogeneity of vegetable seeds. So, it was tested on different quality seeds of five varieties of vegetable beans (Nastena, Magura, Mirobelа, Morena, Bazhenf) obtained in five contrasting environmental and geographical conditions (Moscow, Belgorod, Stavropol, Omsk, Gorki) in 2011-2012. In the future, the technique can be used to improve the quality of digital images of seeds, study of seed diversity, including to improve the control of the breeding process. In addition, it is applicable to study the relationship of the set of morphometric characteristics of seeds and their sowing qualities.


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