DOCSLIB.ORG

Morphological Assessment of Epididymal Sperm in Wistar Rats Using Different Histological Stains

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Morphological Assessment of Epididymal Sperm in Wistar Rats Using Different Histological Stains Short Communication Acta Vet Eurasia 2020; 46: 132-136 Morphological Assessment of Epididymal Sperm in Wistar Rats Using Different Histological Stains Nathan Isaac DIBAL , Sani Hyedima GARBA , Tamunotonye Watson JACKS Department of Human Anatomy, University of Maiduguri, Maiduguri, Nigeria Cite this article as: Dibal, N.I., Garba, S.H., Jacks, T.W., 2020. Morphological Assessment of Epididymal Sperm in Wistar Rats Using Different Histological Stains. Acta Vet Eurasia 2020; 46: 132-136. ORCID IDs of the authors: N.I.D. 0000-0002-1805-7553; S.H.G. 0000-0001-6165-0064; T.W.J. 0000-0002-1096-2997 Abstract Spermatozoa are unique and highly specialized cells operat- blue, Alcian blue, Harris hematoxylin, and eosin stains. The re- ing at the microscopic level in a complex environment and sult showed that the head and the proximal and distal parts have various sizes, shapes, and forms. The sizes, shapes, and of the tail were stained excellently with crystal violet and H&E forms of the spermatozoa are dependent on the species but stains. Giemsa and Harris hematoxylin were also good but may be different even within the same species. Five Wistar rats could not stain the distal part of the tail. In conclusion, crystal were euthanized using ketamine injection. The epididymis of violet is the best stain for sperm morphology in Wistar rats fol- each rat was dissected, teased and diluted in normal saline. lowed by H&E stain. Several smears were prepared and stained with Giemsa, Leish- Keywords: Crystal violet, eosin, hematoxylin, morphology, man, hematoxylin and eosin (H&E), crystal violet, methylene sperm Introduction copy (Sousa et al., 2013; Villaverde et al., 2008). Different stains may exert different effects on the spermatozoa. Therefore, it Spermatozoa are unique and highly specialized cells operating can significantly affect the results of morphometric measure- at the microscopic level in a complex environment and have ment and consequently, the sperm quality (Banaszewska et al., various sizes, shapes, and forms (Gage and Freckleton, 2003). 2015). The structure of the mammalian sperm is designed in The sizes, shapes, and forms of the spermatozoa are depen- such a way that it enables them to penetrate the ovum, giving dent on the species but may be different even within the same them the ability to transfer genetic information in the process species (Andraszek et al., 2018; Banaszewska et al., 2015). The of fertilization (Kondracki et al., 2017). Examination of sperm differences in the sperm sizes and shapes within different spe- morphology allows one to determine the fertility status in a cies, coupled with the fact that spermatozoa are always hetero- male (Phillips et al., 2004). geneous in a single ejaculate (normal and abnormal/damaged sperm), are the major obstacles in proper semen analysis (Ban- Staining of the sperm is important to study the affinity of the aszewska et al., 2015). Understanding the adaptive significance different parts of the spermatozoa to various histological stains. of sperm form and function in different species has been a chal- Several stains are used for sperm in different species, and they lenge to biologists; therefore, various staining techniques have include Giemsa-Wright (Perez-Marin et al., 2016), eosin-nigrosin been developed for different species (Gage, 1998). Sperm stain- (Oridupa et al., 2018), eosin and Leishman (Isaiah et al., 2018), ing is aimed at easy visualization of the cells and providing a eosin-gentian (Kondracki et al., 2017), Weigert’s ferrous hema- better identification of the abnormalities through light micros- toxylin, hematoxylin & eosin (H&E), Papanicolau, orange G, light Address for Correspondence: Nathan Isaac DIBAL • E-mail: [email protected] This work is licensed under a Creative Received Date: 15.04.2020 • Accepted Date: 04.06.2020 • Available Online Date: 23.07.2020 • DOI: 10.5152/actavet.2020.20018 Commons Attribution-NonCommercial Available online at actavet.org 4.0 International License. 132 DIBAL et al. Morphological Assessment of Sperm of Wistar Rats Acta Vet Eurasia 2020; 46: 132-136 Figure 1. Spermatozoa stained with Giemsa at 400x Figure 2. Spermatozoa stained with hematoxylin and eosin magnification at 400x magnification green, Shorr, and Janus green (Aksoy et al., 2012). Microscopic ter ad libitum. The research was approved by the Department evaluation of sperm morphology is relatively simple but can of Human Anatomy ethical committee (Code No. UM/HA/ produce results similar to those obtained with more sophisti- PGR19.20-09900). The rats were euthanized using ketamine in- cated and expensive methods (Buendia et al., 2002). Papanico- jection. The whole epididymis of each rat was dissected, teased, laou staining is time consuming, multistage, and involves more and diluted in 5 mL of 10% normal saline at room temperature. than 10 different solutions (Van der Horst and Maree, 2009). Several smears were prepared on a glass slide, allowed to dry Therefore, the method has a limited applicability in case of at room temperature, and fixed with ethanol. The smears were urgent need. Eosin-nigrosin staining is a popular method for stained with crystal violet stain (C25H30ClN3), methylene blue semen evaluation in mammals and birds (Bjorndahl et al., 2004; stain (C16H18ClN3S), Alcian blue (C56H68Cl4CuN16S4), hematoxylin Lącka et al., 2016). The method enables the analysis of sperm (C16H14O6), eosin Y (C20H6Br4Na2O5), Giemsa stain (buffered solu- viability and evaluation of its structure. Eosin-nigrosin staining tion of methylene blue, eosin Y, and Azure B in methanol and is easy to perform and allows detection of the morphological glycerol), Leishman stain (buffered solution of methylene blue abnormalities as well as determination of membrane integrity and eosin Y in alcohol), and H&E stain. The stains were washed of the sperm. after 5–10 minutes depending on the type of stain used and al- lowed to dry at room temperature. All the slides were observed Animals have been used for scientific studies mainly to develop under light microscope at 400x magnification. a better understanding of animal and human anatomy, physiol- ogy, and pathology (Andersen and Winter, 2019). Domesticated Results rats (Rattus norvegicus) were the first rodent species to be used Photomicrograph of the spermatozoa of Wistar rats showed a in research as early as 1828. In the 20th century, rats became the characteristic sickle-shaped head and a long slender tail. The preferred animal model in research. The first standard rat strain outline of the head and proximal part of the tail were seen (Wistar rat) was developed in 1909, and it is from this strain that clearly with Giemsa stain; both the outline of the head and most of the rats used in laboratories today are believed to have proximal part of the tail were stained blue (Figure 1). The heads descended. Wistar rats are the most frequently used rat model of the spermatozoa were stained red and blue with H&E; the in research (Franco, 2013; Lindsey and Baker, 2006). Therefore, proximal part of the tails was stained dark blue while the dis- understanding the sperm morphology of Wistar rats will pro- tal part was stained pale blue. The central part and outline of vide a clue for studying the sperm cells in different vertebrates, the head appeared red and blue, respectively (Figure 2). Harris including humans. The aim of this study was to assess the hematoxylin and Leishman stains gave an outline of both the morphology of epididymal sperm in Wistar rats using different head and tail of the spermatozoa; the middle part of the head stains. did not pick any stain. The tail appeared light blue with Harris Materials and Methods hematoxylin and dark blue with Leishman stain. The proximal part of the tail stained dark blue while the distal part stained A total of 5 male Wistar rats between 8 and 9 weeks old, weigh- light blue with Leishman stain (Figures 3 and 4). The heads and ing 118–126 g were used for the study. They were fed with tails of the spermatozoa were stained purple with crystal violet. grower mash (Vital Feed, Grand Cereal Jos, Nigeria) and wa- The heads were completely stained, and both the proximal and 133 DIBAL et al. Morphological Assessment of Sperm of Wistar Rats Acta Vet Eurasia 2020; 46: 132-136 Figure 3. Spermatozoa stained with Harris hematoxylin at Figure 5. Spermatozoa stained with crystal violet at 400x 400x magnification magnification Figure 4. Spermatozoa stain with Leishman stain at 400x Figure 6. Spermatozoa stained with methylene blue at 400x magnification magnification distal parts of the tail were also stained (Figure 5). The sperma- of stallion (Serafini et al., 2013). Giemsa stain was reported to tozoa stained with methylene blue and Alcian blue were ob- give a clear head morphology of human spermatozoa, but the served as an outline of the head and proximal part of the tail; middle piece and the tail morphology were not clear (Aksoy et however, the distal part of the tail was not stained even with al., 2012). In this study, H&E showed a good staining of the head prolonged time of exposure to the stains (Figures 6 and 7). The as well as the tail. Earlier studies reported H&E as an excellent heads of the spermatozoa stained with eosin appeared bright stain for human spermatozoa morphology as it could stain the red with a dark outline, the proximal part of the tails stained head, acrosome, and tail with a clear distinction between all the blue, and the distal part of the tail did not pick any stain even parts (Kondracki et al., 2017). after prolonged exposure to the stain (Figure 8). Lingappa et al. (2015) identified the H&E stain as the best one Discussion for the assessment of human sperm head morphology. The stains gave a clear morphology of the head as purple conden- With the Giemsa stain, a clear outline of the sperm can be ob- sation; detailed morphology of the middle part and the tail served, which indicates that this stain is good for the cell mem- were also clearly identified.
Load more

Recommended publications
  • Eosin Staining
    Science of H & E Andrew Lisowski, M.S., HTL (A.S.C.P.) 1 Hematoxylin and Eosin Staining “The desired end result of a tissue stained with hematoxylin and eosin is based upon what seems to be almost infinite factors. Pathologists have individual preferences for section thickness, intensities, and shades. The choice of which reagents to use must take into consideration: cost, method of staining, option of purchasing commercially-prepared or technician-prepared reagents, safety, administration policies, convenience, availability, quality, technical limitations, as well as personal preference.” Guidelines for Hematoxylin and Eosin Staining National Society for Histotechnology 2 Why Do We Stain? In order to deliver a medical diagnosis, tissues must be examined under a microscope. Once a tissue specimen has been processed by a histology lab and transferred onto a glass slide, it needs to be appropriately stained for microscopic evaluation. This is because unstained tissue lacks contrast: when viewed under the microscope, everything appears in uniform dull grey color. Unstained tissue H&E stained tissue 3 What Does "Staining" Do? . Contrasts different cells . Highlights particular features of interest . Illustrates different cell structures . Detects infiltrations or deposits in the tissue . Detect pathogens Superbly contrasted GI cells Placenta’s large blood H&E stain showing extensive vessels iron deposits There are different staining techniques to reveal different structures of the cell 4 What is H&E Staining? As its name suggests, H&E stain makes use of a combination of two dyes – hematoxylin and eosin. It is often termed as “routine staining” as it is the most common way of coloring otherwise transparent tissue specimen.
    [Show full text]
  • Basics of Hematology and Patho-Histology
    Basics of Hematology and Patho-histology Practical Course in Molecular Pathology Winter Term 2015 Ernst Müllner MFPL (Max F Perutz Laboratories) Department of Medical Biochemistry Medical University of Vienna [email protected] www.mfpl.ac.at/mfpl-group/group/muellner.html (Müllner homepage / research) E. coli + macrophages medicalschool.tumblr.com/post/43914024728/sem-image-of-e-coli-bacteria-and-macrophages medicalschool.tumblr.com/post/18256087351/r ed-blood-cells-erythrocytes-trapped-by-fibrin Overview on main white blood cell (WBC) types – (Wikipedia) Mature white blood cell types I White Blood cells (WBCs) are frequently also referred to as peripheral blood mononuclear cells (PBMCs). Granulocytes in general are part of the innate immune system. Names derive from staining with hematoxylin and eosin. Whereas basophils stain dark blue and eosinophils are bright red, neutrophils stain neutral to pink. Basophil granulocytes Eosinophil granulocytes Neutrophil granulocytes Least common granulocyte type About 1-6% of WBCs; component Most abundant WBC type (40- (0.01- 0.3% of WBCs. Large of innate immune system to com- 75%) and essential part of the cytoplasmic granules obscure the bat parasites and certain infec- innate immune system. A patho- nucleus under the microscope. tions; also associated with allergy gen is likely to first encounter a When unstained, the nucleus is and asthma. Following activation, neutrophil. Normally contain a nu- visible and usually has 2 lobes. eosinophils effector functions in- cleus of 2-5 lobes. Neutrophils Basophils appear in inflammatory clude production and release (de- quickly congregate at a infection reactions, particularly those granulation) of cytotoxic substan- site, attracted by cytokines from causing allergies, mainly via the ces (granule proteins, reactive activated endothelium, mast cells, vasodilator histamine (antihistami- oxygen species …) and production or macrophages.
    [Show full text]
  • Induced Osteogenesis in Plants Decellularized Scaffolds
    www.nature.com/scientificreports OPEN Induced Osteogenesis in Plants Decellularized Scafolds Jennifer Lee1,4, Hyerin Jung2,3,4, Narae Park2,3, Sung-Hwan Park1 & Ji Hyeon Ju1,2,3* A three-dimensional (3D) culture system that closely replicates the in vivo microenvironment of calcifying osteoid is essential for in vitro cultivation of bone-like material. In this regard, the 3D cellulose constructs of plants may well serve as scafolds to promote growth and diferentiation of osteoblasts in culture. Our aim in this study was to generate bone-like tissue by seeding pluripotent stem cells (hiPSCs), stimulated to diferentiate as osteoblasts in culture, onto the decellularised scafolds of various plants. We then assessed expression levels of pertinent cellular markers and degrees of calcium- specifc staining to gauge technical success. Apple scafolding bearing regular pores of 300 μm seemed to provide the best construct. The bone-like tissue thus generated was implantable in a rat calvarial defect model where if helped form calcifed tissue. Depending on the regularity and sizing of scafold pores, this approach readily facilitates production of mineralized bone. It is well established that three-dimensional (3D), rather than two-dimensional (2D), culture ofers a microenvi- ronment closer to in vivo conditions, better enabling in vitro development of organoids. Similar with other organs, there is a growing clinical need for bone organoids, which may be particularly suitable substitutes for less available autologous bone grafs, helping to repair critical bony injuries or congenital defects. Unfortunately, current engi- neering techniques involving bone are largely restricted to 3D culture of osteoblasts. Hence, the term ‘bone-like tissue’ seems more apt than ‘bone organoid’.
    [Show full text]
  • Product Name Here Routine Stains & Special Stains
    ® A Division of General Data Healthcare Histology Innovation for a NEW Generation Ready-To-Use Reagents, Dyes & Stains RoutineProduct Stains Name & SpecialHere Stains Single Source For Your Histology Reagents. Protecting Every Life Story In Your Lab. Each tissue specimen your lab processes has a life story behind it. Your mission is to ensure all your specimens receive optimal processing in order to deliver the best possible care. Our product line of histological reagents, dyes and ready-to-use stains enable your lab to deliver increased productivity, advanced specimen safety and provide highly accurate processing and staining results. ROutINE stAIns Eosin Y, 1.0% alcoholic solution, Non-Acidic Cytoplasmic counterstain useful in immuno-histochemistry or treatment of tissue sections with hematoxylin. Contains no acetic acid. Ready-to-use with any automated stainer. Cat. # Description E-1Y1P Eosin Y, 1%, 16 oz/ea; See MSDS for HAZ-MAT handling requirements under transportation on Air Shipments; Store at 25° C E-YP50GR Eosin Y Certified Powder, 50gm/ea. See MSDS for HAZ-MAT handling requirements under transportation on Air Shipments; Store at 25°C Gill’s #2, double strength for Histology & Cytology Used when a stronger or darker nuclear stain is required for cytology or immunohistochemistry (IHC) counterstaining. Gill’s #2 formulation is a double strength mixture, stains darker and more quickly than Gill’s #1. General purpose nuclear stain, progressive type. Used with hematoxylin and eosin staining. Cat. # Description H-G21L Gills 2 Hematoxylin, 1L/ea. See MSDS for HAZ-MAT handling requirements under transportation on Air Shipments; Store at room temp.
    [Show full text]
  • Developing Fluorogenic Reagents for Detecting and Enhancing Bloody Fingerprints
    The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Developing Fluorogenic Reagents for Detecting and Enhancing Bloody Fingerprints Author: Robert M. Strongin, Dr.Martha Sibrian-Vazquez Document No.: 227841 Date Received: August 2009 Award Number: 2007-DN-BX-K171 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally- funded grant final report available electronically in addition to traditional paper copies. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Developing Fluorogenic Reagents for Detecting and Enhancing Bloody Fingerprints Award 2007-DN-BX-K171 Authors Prof. Robert M. Strongin Dr.Martha Sibrian-Vazquez 1 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Abstract Fingerprints are the most common and useful physical evidence for the apprehension and conviction of crime perpetrators. Fluorogenic reagents for detecting and enhancing fingerprints in blood, however, have several associated challenges. For instance, they are generally unsuitable for dark and multi-colored substrates. Luminol and fluorescin and other chemilumigens and fluorigens can be used with dark and often multi-colored substrates, but are not compatible with fixatives and their oxidation products are not insoluble.
    [Show full text]
  • GHS,Hematoxylin Stains Procedure
    HEMATOXYLIN STAINS 4. If eosin staining is excessive, nuclear staining may be masked. Proper eosin staining will (Procedure No. GHS) demonstrate a 3-tone effect. To increase dif feren ti a tion of eosin, extend time in alcohols or use a first alcohol with a higher water content. The times in the alcohols may be _______________________________________________ adjusted to obtain the proper degree of eosin staining. 5. Positive control slides should be included in each run. INTENDED USE 6. The data obtained from this procedure serves only as an aid to diagnosis and should be _______________________________________________ reviewed in conjunction with other clinical diagnostic tests or information. Gill Hematoxylin solutions are nuclear stains intended for use in Histology and Cytology. Hematoxylin Solutions, Gill Nos. 1, 2 and 3 are for “In Vitro Diagnostic Use”. PROCEDURE 1: Hematoxylin, a common nuclear stain, is isolated from an extract of logwood (Staining Exfoliative Cytology Preparations Using Hematoxylin Solution, Gill No. 1 or Gill No. 2) (Haematoxylon campechianum).1 The first successful bi o log ic application of hematoxylin 1. Fix cytologic smears in 95% ethanol..................................................................15 minutes was described by Bohmer1 in 1865. Since then numerous formulations have appeared. Of 2. Rinse in gently running tap water.......................................................................30 seconds these, Harris’, Gill’s, Mayer’s and Weigert’s have retained popularity. Before hematoxylin can 3. Stain in Hematoxylin Solution, Gill No. 1 or Gill No. 2.....................................1.5-3 minutes be used as a nuclear stain, it must be oxidized to hematein and combined with a metallic ion 4. Rinse in tap water. (mordant). Most successful mordants have been salts of aluminum or iron.
    [Show full text]
  • Article Text Additional Text Cinr Schulznr Casnr Item Number
    The whole world of dyes and dye solutions Chroma-products – subdivided in dyes, dye solutions, indicators and auxiliaries article text additional text item number CINr SchulzNr CASNr hazardous Acid blue 119 1B-555 42765 1324-76-1 Carbolic Gentian Violet 2E-028K Y China Blue 1B-507 42755 816 discoloration solution acetone/Ethanol 1:1 E333 Y nuclear fast red solution (0,1%) aqueous 2C-337 6409-77-4 rhodamine b, ethanolic (1%) 2C-339 64-17-5 Y Safranin solution 2C-333K Y shipping cost chroma solvent green 3 1B-553 128-80-3 staining reagent eppendahl II 1A-652 Water Blue TR, Unna 1B-517 42755 816 Dienstag, 28. September 2021 SEITE 1 VON 21 The whole world of dyes and dye solutions Chroma-products – subdivided in dyes, dye solutions, indicators and auxiliaries article text additional text item number CINr SchulzNr CASNr hazardous dyes Acid Alizarine Blue B 1A-252 16680 1058-92-0 Acid Black 12 B 1A-598 20470 299 1064-48-8 acid brilliant flavine 7g 1F-562 61968-07-8 Acid Fuchsine-Orange 1F-347 Acid Green G 1B-215 42095 765 5141-20-8 Acid Rhodamine 1A-004 45100 863 3520-42-1 Acridine Orange 3 R zinc chloride double salt 1B-307 46005 10127-02-3 Acridine Yellow 1B-331 46025 135-49-9 Acriflavine 5A-406 46000 906 Y Alcian Green 2 GX 1F-555 Alcian Green 3 BX 1F-551 Alcian Yellow GXS 1F-597 12840 61968-76-1 Alizarine Blue B 1A-246 16680 Alizarine Brilliant Violet R 1B-077 60730 1196 4430-18-6 Alizarine Carmine 1F-581 58005 1145 130-22-3 Alizarine Pure 1A-020 58000 1141 72-48-0 Alizarine Purple RS 1B-079 60730 1196 4430-18-6 Alizarine Red S 1F-583 58005
    [Show full text]
  • Diagnostic Stains & Laboratory Reagents
    polysciences.com Diagnostic Stains & Laboratory Reagents • Cytology • Dermatology • General Reagents • Hematology • Histology • Microbiology U.S. Corporate Headquarters | 400 Valley Rd, Warrington, PA 18976 | 1(800) 523-2575 (215) 343-6484 | Fax 1(800) 343-3291 | [email protected] Polysciences Europe GmbH | Badener Str. 13, 69493 Hirschberg an der Bergstr., Germany | +(49) 6201 845 20 0 | Fax +(49) 6201 845 20 20 | [email protected] Polysciences Asia Pacific, Inc. | 2F-1, 207 DunHua N. Rd. Taipei, Taiwan 10595 | (886) 2 8712 0600 | Fax (886) 2 8712 2677 | [email protected] Life Sciences Astral Diagnostics Catalog # Size Cytology Acrylic Mounting Medium . 25835-16 16 oz Histo/Cyto Mounting Medium in a toluene matrix using an acrylic resin for coverslipping . 25835-4 4 oz AQUAbluing Reagent . 25836-1 1 gal Bluing reagent used to provide crisp nuclear detail . Bluing Reagent . 25837-32 32 oz Lithium carbonate solution used to provide crisp nucler detail to cells . 25837-1 1 gal Human Colon, 20X Harris Hematoxylin, Eosin Y Pap Stain Gynecological stain used in combination with OG-6 and hematoxylin in the diagnosis of malignant cytological diseases . FDA approved for in vitro diagnostic use . EA-36 25865-32 32 oz 25865-1 1 gal EA-50 25841-2 .5 2 .5 gal EA-65 25842-2 .5 2 5. gal EASYpap . 25843-32 32 oz Gynecological single solution counterstain . A substitute to the traditional Eosin 25843-1 1 gal azure and Orange G . FDA approved for in vitro diagnostic use . Formalin 10%, Buffered . 25848-16 16 oz Most widely used fixative used for routine processing of tissue in histology laboratories .
    [Show full text]
  • The Color of Tissue Diagnostics Routine Stains, Special Stains and Ancillary Reagents
    The Color of Tissue Diagnostics Routine Stains, Special Stains and Ancillary Reagents The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. For over years, 100routine stains, special stains and ancillary reagents have been part of the MilliporeSigma product range. This tradition and experience has made MilliporeSigma one of the world’s leading suppliers of microscopy products. The products for microscopy, a comprehensive range for classical hematology, histology, cytology, and microbiology, are constantly being expanded and adapted to the needs of the user and to comply with all relevant global regulations. Many of MilliporeSigma’s microscopy products are classified as in vitro diagnostic (IVD) medical devices. Quality Means Trust As a result of MilliporeSigma’s focus on quality control, microscopy products are renowned for excellent reproducibility of results. MilliporeSigma products are manufactured in accordance with a quality management system using raw materials and solvents that meet the most stringent quality criteria. Prior to releasing the products for particular applications, relevant chemical and physical parameters are checked along with product functionality. The methods used for testing comply with international standards. For over Contents Ancillary Reagents Microbiology 3-4 Fixing Media 28-29 Staining Solutions and Kits years, 5-6 Embedding Media 30 Staining of Mycobacteria 100 6 Decalcifiers and Tissue Softeners 30 Control Slides 7 Mounting Media Cytology 8 Immersion
    [Show full text]
  • Influence of Histochemical and Immunohistochemical Stains on Polymerase Chain Reaction Takayuki Murase, M.D., Hiroshi Inagaki, M.D., Tadaaki Eimoto, M.D
    Influence of Histochemical and Immunohistochemical Stains on Polymerase Chain Reaction Takayuki Murase, M.D., Hiroshi Inagaki, M.D., Tadaaki Eimoto, M.D. Department of Pathology, Nagoya City University Medical School, Nagoya, Japan longed the CYCLEmin (P < .01), and the PCR ampli- The polymerase chain reaction (PCR) analysis of fication did not reach the “plateau” level with a DNA extracted from tissue sections can be applied maximum of 60 cycles. The PCR efficiency was to a variety of research and diagnostic protocols. To worse in microwave or pressure cooker treatment, analyze selectively the specific areas of tissue, a di- with neither CYCLEmin nor CONCmax being ob- rect microdissection of histochemically or immuno- tained. When target areas from sections for subse- histochemically stained sections, if satisfactory for quent PCR amplification are microdissected, PCR, is helpful. However, the influence of various methyl green is most suitable as a dye for nuclear staining methods on PCR has been poorly investi- staining. The immunohistochemical visualization gated. In this study, paraffin sections of formalin- with DAB or new fuchsin yields no unfavorable ef- fixed lymph node samples were histochemically fects. A successful PCR amplification may not be stained with Mayer’s hematoxylin, eosin Y, methyl expected in sections that are pretreated in a micro- green, or May-Grunwald solution and immuno- wave oven or pressure cooker. stained for CD45 using 3,3؅-diaminobenzidine (DAB), DAB with cobalt ion (DAB-Co), or new fuch- KEY WORDS: Histochemical stain, Immunohisto- sin as the chromogen. In addition, unstained sec- chemical stain, Microdissection, Polymerase chain tions were treated with trypsin, microwave, or pres- reaction, Pretreatment.
    [Show full text]
  • Finger Mark Development Techniques Within Scope of ISO 17025, Sections 1
    Fingerprint Source Book – Chapter 3: Finger mark development techniques within scope of ISO 17025 Chapter 3: Finger mark development techniques within scope of ISO 17025 3.1 Acid dyes (acid black 1, acid violet 17, acid yellow 7) 1. History 1.1 Fingerprints may deposited in a number of contaminants at crime scenes, and of all these blood is the most commonly observed This is possibly because, when present even in small quantities, it is easily seen as it strongly absorbs light throughout the visible spectrum. However, when present in minute amounts, or on dark, patterned or multicoloured confusing backgrounds, the blood may require enhancement to make it more useful for evidential purposes. Additionally, proof that a stain is actually blood rather than an innocuous substance may be important in assessing guilt or innocence, and may even be a matter of life or death in some cases. 1.2 The history of proving the presence of blood evidence in forensic investigation dates back over 150 years using chemical means, and further still when microscopical methods are considered. Anton van Leeuwenhoek was said to be the first person to describe and illustrate blood cells in the latter part of the 17th century, although this is disputed. 1.3 The earliest tests for blood were of two types, both relying on the presence of the haem group present in the red blood cells. The early tests included those that reacted with haem to produce crystals and those that relied on its catalytic nature. More recently (1999) a third test relying on antibodies has been introduced.
    [Show full text]
  • Biosciences T Polysciences, Inc
    Facts Visit Polysciences, Inc. Poly| No. 3 Vol. 3 BioSciences at NSH in Pittsburgh, PA • Fixatives from News l Views l Insights • Embedding Kits • Paraffins Histology • Embedding Molds Professionals IN THIS ISSUE. • Certified Dyes, Stains Are you ready? Page 1 and more Guide to Special Stains . Page 2 Visit Booth #1001-1003 to learn about new products Comparison of Hematoxylin and Eosin Stains on Automated Stainers . Page 3 to help increase productivity in your laboratory and Page 4 Science Solutions: Biosciences Q & A . enter for a chance to win a DVD player! Product Showcase . September 14 - 16, 2008 Guide to Special Stains Polysciences’ special stains are a diverse familyoscope of slide to basedidentify stains various that tissues,rely on chemical reactions for visualization by micr structures, cells and microorganisms. Special stains are an important tool for pathologists in defining a patient’s medical profile.e Clinical key in applications detecting fora specific special stains cover a wide range of diseases and ar pathogen. Polysciences’ special stains are an integral part of the research laboratory and monitoring pathological diseases. B in identifying A tified Powdered Dyes for Visualizing Special Stain Kits and Cer Tissues, Structures, Cells and Microorganisms Visualization Cat. # Size Stain Description Kinyoun Kit Mycobacteria 1 Kit AFB (Acid-Fast Bacteria) 24670 Amyloids 02736 25g Congo Red, C.I. 22120, certified ussian Blue) Ferric iron and ferritin 24199 1 Kit Iron Stain (Pr D 24205 1 Kit Gomori's Trichrome Stain Kit Connective fiber, muscle in tissue C 24668 1 Kit Gram Stain Kit Bacteria 24611 500ml Luxol Fast Blue Mitochondria 08824 100ml Multiple Stain Solution Mast cells 08711 470ml Wright Giemsa Stain Solution Mast cells, bloodMast films,cells parasites , C.I.
    [Show full text]