Practical training in Histology and Embryology Information on practical course ■ Tuesday 10:30 -13:00 ■ RNDr. Petr Vaňhara, PhD (30) ■ Dr. Hana Kotasová, PhD (32) ■ Prof. Miroslava Sedláčková, CSc (33) ■ MUDr. Ivana Baltasová (36) General issues Histology and Embryology Sample processing Organization issues • Beginning - 10:30 strictly • Change your shoes - you will not be allowed to enter the hall w/o indoor shoes • Lockers - Jackets, coats, bags etc. • Cell phone - switched off or in silent mode • Microscopic hall = laboratory - eating, drinking, smoking not allowed - smoking strictly forbidden anywhere in LF - students have to follow the instructions - academic misconducts or inappropriate behavior result in excluding from the lesson or course Follow safety rules You have dedicated working place You are responsible for microscope, slide set, EM atlas Practical lesson Introduction Your individual work = study of the slides, schematic but precise drawing of tissue architecture, careful description. You make your own „study atlas'' Students come prepared for practices - schedules and syllables - pin-boards dpt. webpage Your knowledge is verified during semester Break -10 minutes Attendance 100% attendance Substitution only in exceptional cases, after permissions from both the teach of your group and the lesson where you plan to substitute Sign in to the list Make a protocol, let it check and sign by the lecturer Registration of substitution: Datum Date Jméno Name Ročník Year Skupina Group Č. praktika Nr. of practice Č. místa Nr. of place Vyučující - podpis Teacher- signature Protocols you have to make paper protocols (no tablets, laptops) A4 size, blank, without lines, according to the template pencil hand drawings (no pen) complete set of your protocols is required for getting the credits the quality of the protocol is approved by your teacher's signature at the end of practical lesson Low-quality protocols cannot be approved and you have to substitute the respective practical lesson Testing your knowledge every student is examined 5x per semester testing the knowledge of structures, including their English and Latin names, functions and biological context short written test with images or schemes, results: „Passed" or „Failed" You have to successfully pass all 5 tests If you fail in partial test, you can repeat it once per semester failing in the partial tests result in the overal Credit test at the end of semester 1 Seznam preparátu ke studiu: Číslo název (barvení) Atlas EM: doporučene obrázky ke 5 str. název e Protokol c. Datum:. Jrneno:............................................... Ročník:........................^^^^umna; Pokyny pro vypracování protokolu 1. Student vyhotoví barevné nákresy histologických preparátů (pastelky) nebo černobílé nákresy obrázku z atlasu ejejíjj^njjfiiajnä [obyčejná tužka). 2. Kazdy nákres musí být opatřen následujícími údaji: - název preparátu s uvedením metody barvení fviz Seznam výše}, event. název eJeJ$Jo;0fiEJ3iIlU - zvětšení: 10 k 4/ 10 k ID / 1dx20 / 10 x AQjUx okular x objektiv) nebo ceJt ÍSÍ-: 40x/ 100>; / 200x / 4°°* - popis obrázku. Kontrola protokolu Praktické cvičení: podpis učitele • Credits -100% attendance - complete set of signed protocols from all lessons - Passed five tests • End of practice: 13:00 - The practice is closed by the lecturer i DEPARTMENT OF HISTOLOGY AND EMBRYOLOGY FACULTY OF MEDICINE - MASARYK UNIVERSITY Department of Histology and Embryology Faculty of Medicine MU http://www.med.muni.cz/histoloqy Semester 1, 2013/2014 Programme of lectures and nracticals in histology and embryology for the 1st year of General medicine Lecturers: Prof. MUDr. ENDr. S. Čech. DrSc, Doc. MVDr. A. Hampl, CSc. Seir-insr tutor;: Doc. MUDr. M. Sedláčková. CSc. MUDr. J. Du::iková. MUDr. L. Krejčířovi Ph.D., MUDr. I. Lmisdioyi Ph.D., RKDr. P. Vaňhara, Ph.D. Lectures CĚŽ. H!) 1. IS. 02. - 22. 02. 201? Introduction _ke object and significance of histology. Relevance of histologv to o:her biomedical disciplines. Hisroiv. cuiTenl s:ate, and ful"j.-e c: histologv. Iethodologies to studv a structure Cytology _ke cek - definition, characteristics. Cell nucleus - ultra st--.ic:ure and function. 2. 25.02.-01.03.2013 Organelles - structure, localization, a function. Cell inclusions and oigmem Cvroskele:on - micrcfilame: filamer.ts and microtubules. Cell surface structures and intercellular bonds. Cell division cvcle. Cell differentiator, oek migration, general aspects. 1 04. 03. - OS. 03. 2013 General histology -issues - definition, then origin, and Connective and supporting tissue - geners.1 characteristics, then components and classification. The connective tissue oroper -:ypei. than: distribution, and function. >".iooo.-:inz Tissues - cartuaze and cone - tvoes. mam distribution, ar.o function. Histogenesis 4. 1L 03. - 15. 03. 2013 Epithelial tissue - defimticn. classification, and histogenesis. Eoitkehal membranes and Practices ~ IS. O2.- 22.02.2013 Inti-cd-ji:::c:i, c:-gan:za::c:i ti: i;n~i-?.U: Lectures & Prac treats (CZ, Si) Required knowledge to exam Lectures &. Practkals (CZ, EN) Required knowledge to exam (CZ, EN) Lectures & Practicals - Oral HE (CZ,H() Required knowledge to exam -Oral HE (CZ,, EN) 2. 25.02.-01.03.2013 Cytology Ultrastructure of the cell nucleus during inte.-phase. ...ltiasTiucf.ne of cek o.-ganelles I (mitochondria, the Go^gi apparatus, the endoplasmic reticulum, ribosomes, rysosomes). Aids: At!as of elect-on micro graphs. 3. O4.O3.-0S.03.2013 Ultrastructure of cell organelles II (peroxisemes, and :ke cemricl) Cell inclusions. The arrangement of cell surfaces: aoical, lateral, and casa: ones. Intercellular iunctions: adhering, occluding, and embryo Logy and teratology :. ít3.:-í.:3:t EN: 111742586^8268456^ Recommended literature ľovnh 1 dUhxi Histology a Text and atlas ■ Gh&h l. fünf ■■ ■-■ ._-...■= 'Y Histology A \M~y_'»■■■> A:las Michael H. R055 . Wojriech' Wojríech'PäÁiílina ^/fi^J^ N BITER'S i'li ESSENTIAL rill HISTOLOGY ^ Include* 500 full-color OVERSIZED phatErnlcrogfaphs for use In trie histology laboratory ► SELF-ASSESSMENT section provides 60 test pnolomrcrographs lor identification of tissues and organs with similar morphologic features color atlas of basic histology T i IRWIN BERMAN (LÄNGE! Landman's Medical Embryolo T. W. Sadler JUNQUEIRA _ Basic ■ Histology TEXT & ATLA.5 ¥ A, V Autiioiij^L. Meschcr LANGE Lectures Protocols Ě DEPARTMENT OF HISTOLOGY AND EMBRYOLOGY FACULTY OF MEDICINE - MASARYK UNIVERSITY Home Research Grants Education Publication People Instrumentation Contact Hi n i Hill WMI I 'I Wi' 'fWlíil ilHJi'linín " 'il""T~ i • " í""ľ~n ~ MedAtlas verze 2-í Písmo QOraz Napovedá Obecná histologie Mikroskopická anatomie SSjfflgH^"-'1'' -i.li;> á praktika Master's degree prograr Bad**!"''* <*on"»p oiogr Multimedia, textbooks http://www ► B Text f fšj Atlas ► □ 6.2 Atlas EM snímků ► 0.6.4 Mortotogle ä výyo] l| ► □ 6.5 Embryologie I ► Ü 6.6 Emoryologie II ► □ 6.11 Kardiovaskulární ► Q.6.12L/mfaticltýsystén| ► □ 6.13 Dýchací systém ► □ 6 14 Trávicí systém ▼ & 6.15 Močový systém J3 Ren - přehled S3 Ren - corpusculum gl Ren-cortex 3 Ren - medulla ~) Ren-vazivo 3 Calyx renalis El Ureter-přehled H Ureter-vazivo 3 Vesica urlnalls-tui Urethra feminina-1 3 Urethra masculina || Urethra masculina ► Ů .6 16 Pohlavní system ► □ 6.17 EndoKnnniilázy ► Ö .6 18 Nervový systém *■ □ 6.19 Smyslové orgáni Atlas - Our i interaktivní embryologický atlas clo Ustav histoiogiE a embryológie — Lékařská fakulta MUDr. Jana D u m k o v ä Pt/dékDuäní Zajímavé odkazy ^8 - Vývoj endokrinních žláz T 8-3 Zárodek člověka (8. týden) - vývoj štítné žlázy a příštítných žláz, příčný řez krční krajinou, HE, zvětšení 50x 5 Na celou obrazovku Q|l Skrýt popisky vyzkoušejte se Epitel základů štítné žlázy vytván zpočátku solidní' neluminizované trámce, které se ukládají v dolní' části chrupavky štítné. Z nich se později vytvářejí Folikuly typické stavby. Dorzálné se ke štítné žláze připojují drobná příštítná tělíska. MUDr. Jana Dumkova * I Ostav histologie a embryológie, Lékařská fakulta, Masarykova univerzita ' i i . : : r, j _ . .i, ■;' .u ■■■< ^b■_; - . .'-l n I ■*J5\/ I T = -1. r .i: sspoluprace: ■Až/V I Fakulta informatiky Masarykovy univerzity, 2013 HISTOLOGY • structure and ultrastructure of normal cells and tissues, • cytology and general histology • special histology = microscopic anatomy of individual organs • relevance: oncology, surgery, hematology, pathology, forensic,... EMBRYOLOGY - prenatal (intra uterine) development • General embryology (until 2nd month - EMBRYO ) gametogenesis and early embryonic development • Special embryology (since 3rd month to birth - FETUS ) organogenesis • Teratology - defects in organ development, malformations, anomalies; prenatal screening - ultrasonography, amniocentesis, genetic and karyotype screening • Relevance: gynecology and obstetrics, pediatrics, assisted reproduction / Histology and ulstrastructural architecture / Quantitative analyses of cells and tissues Genomics Transcriptomics Proteomics Metabolomics Physiology Molecular biology Unders Perso Regenerative medicne Assisted reproduction Gene therapy ancer tryerapy Biomedical research Histology cannot be put out of Biochemistry the BW^^Fcal anc nd functional context Histology • Resolution of naked eye - 0,1 mm • Resolution of light microscopy - 10 nm • Resolution of electron microscopy-0,1 nm Tissue processing for the light microscopy (LM) (making of permanent preparations - slides) • SAMPLING (obtaining of material - cells, tissue pieces) • FIXATION of samples (tissue blocks) • RINSING (washing) of samples • EMBEDDING of samples - embedded blocks • CUTTING of blocks - sections • AFFIXING of sections • STAINING of sections • MOUNTING of sections SAMPLING • A small piece of organ (tissue) is sampled and quickly put into the fixative medium. • Biopsy during surgical dissection of organs in living organism = excision = puncture (liver or kidney parenchyma, bone marrow) = curettage (uterine endometrium, adenoid vegetation) • Necropsy from dead individual (sections); in experiments laboratory animals are used and tissue have to be sampled as soon as possible after the break of blood circulation • The specimens shouldn't be more than 5-10 mm3 thick and fixation should follow immediately. FIXATION • Definition: denaturation and stabilization of cell proteins with minimum artifacts) • The reason of fixation: freshly removed tissues are chemically unstable - dry, shrink, undergo hypoxia, autolysis and bacteriological changes • To stop or prevent these changes and preserve the structure tissue samples have to be fixed. During the fixation, all tissue proteins are converted into inactive denaturized (stable) form. 3 main requirements on fixatives: - good preservation of structure - quick penetration into tissue block - no negative effects on tissue staining • Fixatives: solutions of different chemicals - organic fixatives - ALDEHYDES - formaldehyde (most frequently used for LM) -glutaraldehyde (used for EM) - ALCOHOLS - 96 - 100 % (absolute) ethylalcohol - ORGANIC ACIDS - glacial acetic acid, picric acid, trichloracetic acid - inorganic fixatives - INORGANIC ACIDS - chromic acid, osmium tetraoxide (Os04) - SALTS OF HEAVY METALS - mercuric chloride HgC,2 - compound fixatives - mixtures (two or more chemical components to offset undesirable effects fo indiviual (simple) fixatives. FLEMMING's fluid - with Os04 ZENKER'S and HELLY's fluid, SUSA fluid - with HgCI2 BOUIN's fluid - with picric acid CARNOY's fluid - with alcohol Performance: fixatives are carried out at room temperature, the duration varies between 12 - 24 hours, specimen must be covered by 20 - 50 times fixative volume: Ratio of tissue block volume to fixative volume 1 cm3: 20 - 50 cm3 RINSING and EMBEDDING • All samples should be washed to remove the excess of fixative; the choice of rinsing medium is determined by type of fixative: running tap-water or 70-80% ethanol • Relevance of embedding: tissues and organs are brittle and unequal in density, they must be hardened before cutting Embedding media ■ water soluble - gelatine, celodal, water soluble waxes ■ anhydrous - paraffin, celoidin EMBEDDING into PARAFFIN • dehydration - to remove water from fixed samples by ascending series of ethanol is used (50%, 70%, 90%, 96%. each step -2-6 hours • clearing - the ethanol must be replaced with organic solvatant that dissolves paraffin - benzene or xylene • infiltration - melted paraffin wax (56°C) is used; 3x6 hours. • casting (blocking out) - moulds (plastic, paper or metal chambers) are used for embedding. - The moulds are filled with melted paraffin, tissue samples are then placed inside and immediately immersed in cold water to cool paraffin quickly down. - These paraffin blocks are ready for trimming Automated device for tissue dehydration Paper chambers - metal CUTTING Microtome — a machine with automatic regulation of section thickness: 5-10 |im is optimum. sliding microtome - block is fixed in holder, knife or razor moves horizontally rotary microtome - knife is fixed, block holder moves vertically Freezing microtome (cryostat) = rotary microtome housed in freezing box (- 605 c) Cutting of frozen tissue without the embedding Par&ffin 'ribborV Paraffin 'block' AFFIXING Mixture of glycerin and egg albumin or gelatin Section are transferred from microtome razor or knife on the level of warm water (459 C), where they are stretched; then they are put on slides coated with adhesive mixture; excess of water is drained and slides are put in incubator (thermostat, 379 C) over night to affixing of sections. Stretching of sections on warm water STAINING • Different cell or tissue structures are not apparent without staining. • Cellular structures exhibit different affinity to staining dyes alkaline dyes (basic or nuclear) - react with anionic groups of and tissue components basophilia - basophilic structures in the cell acid dyes (cytoplasmic) - react with cationic groups acidophilia - acidophilic structures in the cell neutrophilia - no reaction HE - the most frequent used method ROUTINE STAINING with HEMATOXYLINE -EOSIN (HE) Hematoxyline - basic (nuclear) dye Eosin - acid (cytoplasmic dye • Staining procedure: • paraffin must be removed (dissolved) by xylene • sections are rehydrated in descending series of ethanol (100% ->96% ->80%) • staining with hematoxyline • differentiation in acid ethanol and water (excess of dye is removed) • staining with eosin • rinsing in water (excess of dye is removed) • dehydration in graded ethanol series (80% ->96% ->100%) • clearing in xylene HEMATOXYLINE - EOSIN (HE) Deparaffination Rehydration Washing Staining Differentiation Xylen IV xylen III 100% 96% H20 eosin H20 ethanol ethanol 096885 Staining results: • HE = Hematoxyline - Eosin nuclei - bright clear blue or dark violet cytoplasm and collagen fibers - pink muscle tissue - red • HES = Hematoxyline - Eosin - Safron connective tissue - yellow • AZAN = AZocarmin - ANiline blue - orange G nuclei - red erythrocytes - orange muscle - red collagen fibers - blue Automatic slide stainer MOUNTING • Finally, preparates are closed with coverslip (coverglass) to form a permanent preparáte, bmall amount of mounting medium must be placed between stained section and the coverslip. • Mounting media: soluble in xylene - Canada balsam soluble in water - glycerin-gelatine, arabic gum Permanent histological slides for study in the light microscope Hematoxyline and eosin (HE) Hematoxyline and eosin (HE) 'u % 'V'* cell cytoplasm cell nuclei collagenous connective tissue Hematoxyline and eosin (HE) basophilic cytoplasm of glandular cells (contains ribosomes with RNA) acidophilic cytoplasm of epithelial cells Hematoxyline, eosin and saffron (HES) Collagenous fibers of connective tissue are yellow after staining with saffron Azocarmine and aniline blue (AZAN) Kidney - collagen connective tissue Impregnation of tissue with silver Lien - reticular fibers Cerebellum - nerve fibers Iron hematoxyline Skeletal muscle cells (fibers) Iron hematoxyline Mitochondria in hepatocytes Histochemistry and Immunohistochemistry • Relevance: various chemical compounds detected „in situ" (proteins, AA, NA, saccharides, lipids, enzymes, pigments, inorganic substances - Fe, Ca, Zn) Various epitopes detected by immunotechniques Enzyme conjugated with secondary Ab -visualization Secondary Ab specific against primary Ab Primary Ab specific against epitope of the particular antigen Antigen Actin (cytoskeleton) DAPI (nucleus) Microtubules (cytoskeleton) - Fluorescence labelled proteins Tissue processing for the EM • pH of all solutions (media) must be buffered on 7.2 - 7.4 Cacodylate or phosphate buffer is frequently used. Absolutely dustfree environment • Solutions (media) have to be precise (artifacts) H3C .AS O ll HO"^0\K+ O" K O 11 HO' i"0" Na+ O" Na+ Tissue processing for the EM SAMPLING - immediatelly after arresting of blood circulation, tissue block sized no more than 1mm3 FIXATION - glutaraldehyde (binds amine groups) + Os04 (binds lipids) are used as double fixation RINSING-distilled water DEHYDRATION - ethanol EMBEDDING - gelatin capsule or plastic forms are filled with some medium (which can be polymerized from liquid to solid form) and pieces of fixed tissue are placed into this medium. Epoxyd resins (Epon, Durcupan, Araldite) are usually used as in water insoluble media. CUTTING - ultrathin sections (in ultramictomes) CONTRASTING ~ staining Embedding tools: gelatin (1) or plastic (2) capsules capsule holder (3) embedding plates (4, 5) Embedded blocks prepared for cuttin Trimming the Specimen Block By trimming, using ultramicrotome, an excess of hard medium is removed and pyramide with minimal cut surface (0.1 mm2) is prepared. Minimum of tissue (black) is in the top of pyramid Pyramid Side Profile Too Steep-Trans Am Building (not rigid-vibrations) Too Flat-Pyramid of the Sun (section size changes rapidly) _ \ parallel fust Right-Egyptian Pyramid with, top cut off Cutting Ultrathin sections (70 - 100 nm) -ultramicrotomes. Glass or diamond (b) knives with water reservoir are used Sections slide flow on water in small container attached to the knive Supporting grids KNIFE EDGE CONTRASTING (^STAINING) • principle of differentiation of structures - different dispersion of beam of electrons depending on atomic weight of elements. „electron dyes'' are thus mixtures of heavy metals: uranylacetate or lead citrate stain droplet with floating grid placed section-side down on th droplet Differences between LM and EM LM EM Sampling < 1 cm3 minutes < 1 mm3 seconds Fixation formaldehyde 12-24 hours glutaraldehyde 1-3 hours Embedding paraffin epoxid resins (Durcupan) Cutting Thickness of sections microtome 5-10 |jm Ultramicrotomes 50 - 100 nm Staining (LM) contrasting (EM) dyes {hematoxyline - eosiri) heavy metals (uranylacetatejead citrate) Mounting (only LM) — Result histological slide (preparáte) photograph of ultrathin section Visit us at http://www.med.muni.cz/histoloav DEPARTMENT OF HISTOLOGY AND EMBRYOLOGY FACULTY OF MEDICINE - MASARYK UNIVERSITY Home Research Grants Education Publication People Instrumentation Contact Thank you for attention RNDr. Petr Vanhara, PhD. pvanhara@med. muni, cz