Basic staining methods in histology How to use a light microscope STAINING Different cell or tissue structures are not apparent without staining. Cellular structures exhibit different affinity to staining dyes: alkaline dyes – react with anionic structures BASOPHILIA (basophilic structures in the cell – nucleus, nucleolus, ribosomes, cytoplasm with rough ER) acid dyes – react with cationic groups ACIDOPHILIA – (acidophilic structures in the cell – cytoplasm, smooth ER) no or weak reaction NEUTROPHILIA chromophilic (chromatophilic) x chromophobic polychromatophilic – affinity to both types of dyes xylen I xylen II 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 ROUTINE STAINING with 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 Automatic slide stainer staining set of boxes with media TYPES of STAINING • routine – HE (visualization of all tissue components), Masson´s trichromes: yellow - HES, blue - AZAN, green trichrome (collagen fibers) • special – visualization of particular structures only • orcein, aldehyde fuchsin, resorcin fuchsin - elastic fibres • PAS – polysaccharides • oil red, sudan black – lipids • Schmorl staining • impregnation – AgNO3 - nerve or reticular fibers AFFIXING • attachment of sections by albumin/gelatin to microscopic glasses  permanent slide Hematoxyline Eosin (HE) Results of staining: cell nuclei – blue/violet cytoplasm – pink collagen fibers – pink muscle cells – dark pink/red 2 – Apex linguae (HE) Hematoxyline Eosin Saffron (HES) Results of staining: cell nuclei – blue/violet cell cytoplasm – pink collagen fibers – yellow yellow Masson trichrom 11 – Oesophagus (HES) Azokarmin Aniline blue Orange G (AZAN) Results of staining: cell nuclei – purple cell cytoplasm – pink collagen fibers – blue erythrocytes – orange blue trichrome 99 – umbilical cord (AZAN) Impregnation with AgNO3 Slide 68 – lien Staining – impregnation Result – black reticular fibers Impregnation with AgNO3 Slide 77 – cerebellum Staining – impregnation Result – black nerve processes Orcein Slide 28 – elastic cartilage Staining – orcein Result – red-brown elastic fibers Weigert-van Giesson Slide 31 – renal cortex Staining – Weigert-van Giesson Result – cherry-red collagen fibers Best carmine Slide 49 – vagina - glycogen Staining – Best carmine Result – darc pink glycogen Heidenhain Slide 65 – myocardium Staining – Heidenhain Result – black cardiomyocytes (cross- striation) Schmorl Slide 95 – bone Staining – Schmorl Result – rusty brown bone tissue APPEARANCE OF 3D OBJECTS IN 2D SECTIONS pointer! Obj. 100x / don‘t use it! stage clip holds the slide on stage 9 – use only when using objective 4x The slide must lie on the stage (5) of LM so that the cover glass faces up to the objective lens. Light microscope Light microscope • Eyepieces • Objective lens • Stage with speciment holder • On/Off • Light control • Condenser and Iris aperture • Stage controls • Coarse focus • Fine focus • Light source with diaphragm pointer! obj. 100x / don‘t use it! stage clip holds the slide on stage use only when using objective 4x Rule 1: At the beginning of each practice, check the set of slides and any defects (missing or broken slide) report to teacher. Rule 2: Only one slide can be taken out of the box and studied in LM. Rule 3: The slide must lie on the stage of LM so that the cover glass faces up to the objective lens. Rule 4: Treat the slides carefully; in case of damage of slide, inform the teacher. Rule 5: At the end of each practice, the box with slides must be open for inspection and student must wait at workplace during inspection. HOW TO HANDLE A SET OF SLIDES • Turn on the light. • Start with the 4x objective. • Put the slide on the stage – cover glass must be above • Look through the scope and focus. Use the coarse focus knob at first, until the image is more or less in focus; then switch to the fine focus. • Adjust the light. Not too bright, not too dim. • Adjust the oculars. • Switch to the 10x objective. A slight adjustment with the fine focus knob should get it just right. If you lose the focus and can't see your specimen at all, go back to the 4x and start again. • Switch to the 40x objective if you want to see more detail. Don't use the 100x! • When you want to look at a new slide, switch back to the 4x before changing slides. • Only one slide is out of the box at the moment! Do not remove more! • When you're done with the scope, switch to the 4x and turn the light all the way down before turning it off. • At the end of lesson, the box with slides is checked in your presence before you leave your place Instructions Basic staining methods in histology Slides: • HE staining: 2 – Apex linguae • HES staining: 11 – Oesophagus (37. Epididymis) • AZAN staining: 99 – Funiculus umbilicalis (21. Hepar) • Impregnation: 77 – Cerebellum • (Orcein: 28 – Epiglottis)