Practical training in Histology and Embryology Organization issues •Beginning - strictly on time •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; the images aree available through MedAtlas -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 or dpt. webpage -your knowledge is verified during semester -break – 10 minutes • •Attendance -100% attendance -substitution only in exceptional cases, after permissions from both the teacher of your group and the lesson where you plan to substitute -sign in to the list -make a protocol, let it check and signed by the lecturer Registration of substitution: Datum Jméno Ročník Skupina Č. praktika Č. místa Vyučující - podpis Date Name Year Group Nr. of practice Nr. of place Teacher- signature •Protocols -you have to make paper protocols (no tablets, laptops) -A4 size, blank, without lines, according to the template (can be downloaded from www.med.muni.cz/histology - Education) -(color)pencil handdrawings (no pen) -complete set of signed 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 -incomplete or low-quality protocols cannot be approved and you have to substitute the respective practical lesson • •Testing your knowledge -every student is examined 4´ per semester -testing the knowledge of structures of the previous practical lesson, including the theory (their English and Latin names, functions, development and biological context) AND the theory for the curent practical lesson -short written test with images or schemes, results: „Passed“ or „Failed“ -all images and schemes are made public (MedAtlas, IS) -you have to successfully pass all 4 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 •Credits -100% attendance -complete set of signed protocols from all lessons -passed four tests • •End of practical lesson: -the practice is closed by the lecturer -you are allowed to leave your working place only after checking the microscope and slides -if you leave before the check you may be responsible for any damages/losses recognized later http://www.med.muni.cz/histology Department of Histology and Embryology Faculty of Medicine MU RECOMMENDED LITERATURE Čech, S. a D. Horký. Přehled obecné histologie. 1. vyd. Brno: Masarykova univerzita, 2005. 140 s. ISBN 8021038543. Horký, D. a S. Čech. Mikroskopická anatomie. 2. nezm. vyd. Brno: Masarykova univerzita, 2005. 353 s. ISBN 802103775X. Čech, S., D. Horký a M. Sedláčková. Přehled embryologie člověka. 1. vyd. Brno: Masarykova univerzita, 2011. 187 s. ISBN 978-80-210-5414-1. Mescher, A.L. Junqueira's basic histology :text and atlas. 13th ed. New York: McGraw-Hill Medical, 2013. xi, 544. ISBN 9781259072321. Moore, K.L., T.V.N. Persaud a M.G. Torchia. The developing human :clinically oriented embryology. 9th ed. Philadelphia, PA: Saunders/Elsevier, 2013. xix, 540. ISBN 9781437720020. Vacek, Z. Embryologie :učebnice pro studenty lékařství a oborů všeobecná sestra a porodní aistentka. 1. vyd. Praha: Grada, 2006. 255 s. ISBN 9788024712673. Sadler, T.W. Langmanova lékařská embryologie. 1. české vyd. Praha: Grada, 2011. xviii, 414. ISBN 9788024726403. Kapeller, K. a V. Pospíšilová. Embryológia človeka: učebnica pre lekárske fakulty. Martin: Osveta, 2001. 370 s. ISBN 80-8063-072-0. Lüllmann-Rauch, R. Histologie. Translated by Radomír Čihák. 1. české vyd. Praha: Grada, 2012. xx, 556. ISBN 9788024737294. Ovalle, W.K., P.C. Nahirney a F.H. Netter. Netter's essential histology. 2nd ed. Philadelphia, PA: Elsevier/Saunders, 2013. xv, 517. ISBN 9781455706310. Young, B. Wheater's functional histology :a text and colour atlas. 5th ed. [Oxford]: Churchill Livingstone, 2006. x, 437. ISBN 044306850X. Sadler, T.W. a J. Langman. Langman's medical embryology. Illustrated by Jill Leland. 11th ed. Baltimore, Md.: Lippincott William & Wilkins, 2010. ix, 385. ISBN 9781605476568. Lowe, J.S. a P.G. Anderson. Stevens and Lowe´s Human Histology. 4th. : Elsevier, 2015. ISBN 978-0-7234-3502-0. Lectures Protocols + http://www.med.muni.cz/histology 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 Cell biology Molecular biology Physiology Biochemistry Gene, cell and tissue engineering Quantitative analyses of cells and tissues -Genomics -Transcriptomics -Proteomics -Metabolomics -… Understaning of the whole system -Personalized medicine -Regenerative medicne -Assisted reproduction -Gene therapy -Cancer therapy -Biomedical research - Histology cannot be put out of the biological and functional context Histology •Resolution of naked eye – 0,1 mm •Resolution of light microscopy – 10 nm •Resolution of electron microscopy – 0,1 nm frozensections 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 (OsO4) • – SALTS OF HEAVY METALS – mercuric chloride HgCl2 • - compound fixatives – mixtures (two or more chemical components to offset • undesirable effects fo indiviual (simple) fixatives. • FLEMMING´s fluid – with OsO4 • ZENKER´s and HELLY´s fluid, SUSA fluid – with HgCl2 • BOUIN´s fluid – with picric acid • CARNOY´s fluid – with alcohol • • Fixation is carried out at the room temperature, the time varies between 12 – 24 hours, specimen must be overlayed 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; 3 x 6 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 autotechnikon Leica Automated device for tissue dehydration Leica TP 1020 http://www.schuco.co.uk/images%5Cembedding-moulds.jpg http://www.emsdiasum.com/microscopy/products/histology/imaages2/62527__6.jpg Tissue-Tek® Embedding Rings Tissue-Tek® Process/Embedding Cassette http://www.cmhd.ca/enu_mutagenesis/assets/kidney_tissue_block.jpg Paper chambers - metal CUTTING •Microtome – a machine with automatic regulation of section thickness: 5 – 10 μm is optimum. groot_acc_11 rotary sliding microtome – block is fixed in holder, knife or razor moves horizontally rotary microtome – knife is fixed, block holder moves vertically ERM-230L ESM-150S Rotary microtome Sliding microtome fig3b_2 Freezing microtome (cryostat) = rotary microtome housed in freezing box (– 60º C) Cutting of frozen tissue without the embedding Type-Freezing-Microtome botany_histology7 Cutting a slice of mussel Tweezers drop a piece of mussel into a dehydration cassette Several yellow dehydration cassettes lying in a beaker containing (clear) ethanol Shaving thin slices using a microtome Microtome shaving a slice off a wax block A metal basin filled with warm water Slipping a slide under wax floating in a water bath 1 2 3 4 5 6 7 sampling fixation fixation cutting cutting section affixing section affixing oyster_blockribbon_brush image002 http://www.siumed.edu/~dking2/bluehist/epithel3.gif AFFIXING • •Mixture of glycerin and egg albumin or gelatin • •Section are transferred from microtome razor or knife on the level of warm water (45º 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, 37º C) over night to affixing of sections. HIST005 Medite TFP 40 RTB Stretching of sections on warm water Stretching on a warm plate 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 cell 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 Staining methods: routine – HE, AZAN (demonstrate all components of tissue) special visualizes only special structures impregnation by silver salt for detection of nerve or reticular fibers N_UR_KD_06 Masson-Trichrome-g HistologySlidesOriginal004 DSCN0909 HE – the most frequent used method 1144-3i Lipid droplets detected by oil red 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% Ò96% Ò80%) •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% Ò96% Ò100%) •clearing in xylene vertical staining jar http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif http://www.aname.es/microscopia/ems/histology/HIST44.gif Xylen I XylenII 100% 96% H2O hematoxyline acid ethanol ethanol ethanol Xylen IV xylen III 100% 96% H2O eosin H2O ethanol ethanol HEMATOXYLINE – EOSIN (HE) Deparaffination Rehydration Washing Staining Differentiation Clearing Dehydration Washing Staining Washing 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 A hand holding a slide with a red-stained sample on it mhe_staining tray_of_mhe_oyster_slides vertical staining jar cuvette http://homer.hsr.ornl.gov/CBPS/Arraytechnology/WashSTD.jpg flask http://www.bio-optica.it/gfx/set%203.jpg slides holder (basket) Staining tools: Automatic slide stainer SHANDON LIPSHAW VARISTIAN 24-4 vertical staining jar staining set of boxes with media MOUNTING •Finally, preparates are closed with coverslip (coverglass) to form a permanent preparate. Small 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 http://www.canemco.com/images5/4470.jpg http://nationaldiagnostics.com/images/h1_7a.gif 275496597_bc08febb4b Hematoxyline and eosin (HE) uri04 Hematoxyline and eosin (HE) N_UR_KD_09 cell cytoplasm cell nuclei collagenous connective tissue Hematoxyline and eosin (HE) • 74 basophilic cytoplasm of glandular cells (contains ribosomes with RNA) acidophilic cytoplasm of epithelial cells Hematoxyline, eosin and saffron (HES) • 109 cartillage Collagenous fibers of connective tissue are yellow after staining with saffron Azocarmine and aniline blue (AZAN) • • aza_005 112 Kidney – collagen connective tissue Impregnation of tissue with silver • • 6 Lien - reticular fibers 197%20cortex Cerebellum – nerve fibers Iron hematoxyline • • 22 suppl Skeletal muscle cells (fibers) Iron hematoxyline 101 Mitochondria in hepatocytes MASSON%201 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 Immunohistochemistry Antigen Primary Ab specific against epitope of the particular antigen Secondary Ab specific against primary Ab Enzyme conjugated with secondary Ab - visualization tumblr_ltdcicWc2r1r55lbko1_500 Actin (cytoskeleton) DAPI (nucleus) Microtubules (cytoskeleton) g000457 Ki67BarrettHighgrade 9449_colonc_jp KI-67 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) di-potassium-phosphate-anhydrous-250x250 ANd9GcRuoqAzi1kiMKLoAInGRyc1ZGp45n6J46v9Jp98WUCt9K0OgqcwaA PC34358 Tissue processing for the EM •SAMPLING – immediatelly after arresting of blood circulation, tissue block sized no more than 1mm3 •FIXATION – glutaraldehyde (binds amine groups) + OsO4 (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 http://www.erawat.com/gifs/tablets.jpg BEEM* Capsule Holders Capsules; Embedding, Size 00 Mold for Long Tissues Flat Embedding Molds http://stoopidsavant.com/v-web/gallery/albums/em/IMG_8449.sized.jpg Embedding tools: gelatin (1) or plastic (2) capsules capsule holder (3) embedding plates (4, 5) Embedded blocks prepared for cutting 1 2 3 4,5 microtome Image5 Image6 http://www.udel.edu/Biology/Wags/b617/micro/micro21.gif 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 http://www.udel.edu/Biology/Wags/b617/micro/micro9.gif fig7 Molybdenum MO TEM grids Molybdenum MO TEM grids square transmission Molybdenum MO TEM grids oval transmission Image17 microtome_close_up1 MAN-01 Cutting 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 05-tem-ultramicrotome_zoom ultra_detail 06-tem-ultramicrotome-detail_zoom http://bomi.ou.edu/bmz5364/making-knives_files/image006.jpg https://www.shop.boeckelerdirect.com/images/product_17_lg.jpg Ultramicrotom knives: glass diamond 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 the droplet http://www.med.muni.cz/histol/MedAtlas_2/img_1024x768/HP_img6-2-9.jpg Golgiho aparát ve žlázové buňce Cisterny Granulární ER s transportními váčky Nezralá sekreční granula 24245 Dscn1204 30374 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 mm Ultramicrotomes 50 – 100 nm Staining (LM) contrasting (EM) dyes (hematoxyline – eosin) heavy metals (uranylacetate,lead citrate) Mounting (only LM) --- Result histological slide (preparate) photograph of ultrathin section Visit us at: http://www.med.muni.cz/histology Thank you for attention