INTRODUCTION TO HISTOLOGICAL TECHNIQUES AND SAMPLE PREPARATION FOR LIGHT AND ELECTRON MICROSCOPY DHE MF MU, 2016 Histology • Resolution of naked eye - 0,1 mm • Resolution of light microscopy-0.5 pirn (usually) • 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 various 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 HgCl2 - 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 CUTTING • Microtome - a machine with thickness: 5-10 u.m is optimum. sliding microtome - block is fixed in holder, knife or razor moves horizontally utomatic regulation of section rotary microtome - knife is fixed, block holder moves vertically Sliding microtome Rotary microtome Freezing microtome (CfyOStdt) = rotary microtome housed in freezing box (- 605 c) Cutting of frozen tissue without embedding AFFIXING • Mixture of glycerin and egg albumin or gelatin • Section are transferred from microtome razor or knife on the level of warm water (45Q 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, 37^ 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 Staining methods: routine-HE, AZAN (demonstrate all components of tissue) special visualizes only special structures Lipid droplets detected by oil red HE - the most frequent used method impregnation by silver salt for detection of nerve or reticular fibers Rh» ROUTINE STAINING with HEMATOXYLINE - EOSIN (HE) ■ • 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 - basic (nuclear) dye Eosin - acid (cytoplasmic dye HEMATOXYLINE - EOSIN (HE) Deparaffination Rehydration Washing Staining Differentiation Xylen I Xylenll 100% 96% ethanol ethanol H20 hematoxyline acid ethanol Clearing Dehydration Washing Staining Washing Xylen IV xylen III 100% 96% ethanol ethanol 062991 ^5998895 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 = AZocormin - ANiline blue - orange G nuclei - red erythrocytes - orange muscle - red collagen fibers - blue MOUNTING • Finally, preparates are closed with coverslip (coverglass) to form a permanent preparáte. 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 Hematoxyline and eosin (HE) Hematoxyline, eosin and saffron (HES) f cartillage Q 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 Iron hematoxyline Skeletal muscle cells (fibers) Green trichrome 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 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) HBC* O II HO'^0'K+ O" K O ii HO'^O'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 gelatin (1) or plastic (2) capsules capsule holder (3) embedding plates (4, 5) 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 the droplet Differences between LM and EM LM EM Sampling Fixation Embedding < 1 cm3 minutes formaldehyde 12-24 hours paraffin < 1 mm3 seconds glutaraldehyde 1-3 hours epoxid resins (Durcupan) Cutting Thickness of sections Staining (LM) contrasting (EM) microtome 5- 10 u.m dyes (hematoxyline - eosin) Ultramicrotomes 50-100 nm heavy metals (uranylacetatejead citrate) Mounting (only LM) Result histological slide (preparáte) photograph of ultrathin section