Methods to study Histology

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CellTissueOrgan Organ systemHomeostasis. Histology. How to get tissues for studySteps in tissue preparationFresh tissues from the body1. fixation Formalin ( 10% formaldehyde)Osmium tetroxide for EMMechanism - Forms cross links with proteins (Lysine)2. Embedding gives support for tissue

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Methods to study Histology

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1. Methods to study Histology

2. Cell Tissue Organ Organ system Homeostasis Histology

3. How to get tissues for study Steps in tissue preparation Fresh tissues from the body 1. fixation Formalin ( 10% formaldehyde) Osmium tetroxide for EM Mechanism - Forms cross links with proteins (Lysine) 2. Embedding – gives support for tissue slicing Paraffin or plastic resin 3. Washing & dehydration (dehydration by graded alcohols in ascending order) 4. clearing – to remove paraffin & alcohol By xylol or tulol 5. block making How to get the histology slides?

4. 6. section cutting – 5-10΅ thick sections with microtome 7. mounting – on glass slide ( adhesive – albumin) 8. clearing – xylol / tulol 9. rehydrate – alcohols in descending order Staining nuclear stain – Hematoxylin ( basic stain & water soluble) counter stain – Eosin ( less water soluble but soluble in alcohol) – dehydrate in ascending order 10. Clearing – xylol / tulol 11.Mounting medium – cover glass How to get the histology slides?

5. Staining – routine stain – H&E Some structures are seen/ preserved (large molecules like nucleoproteins, cytoskeleton proteins, ECM proteins- collagen, membrane proteins) some are not seen/lost (small molecules -t-RNA, large molecules like glycogen & Proteioglycans are dissolved, )during the fixation/staining process Special fixatives to retain membrane ( phospholipids) Permanganate & osmium – for EM For Elastic fibers – Orcein/ Resorcin – Fuscin For reticular fibers – Silver impregnation Histochemistry & Cytochemistry Specific binding of dye with particular molecule Fluorescent dye labeled antibody to cell component Enzyme activity Autoradiography – radio isotopes tagged with precursors of a molecule ? molecule incorporated into cell/ tissue before fixation Special situations

6. Basis of staining

7. Mostly resembles basic dye but it is a mordant (helps to form links between tissue fragment & the dye) It will not dissociate in sequential staining process ? unlike other basic dyes What is special about Hematoxylin?

8. What is it ? ? Absorb certain wavelength of light and emit different wavelength Why Metachomasia ? ? Polyanions of tissues bind with dye molecules result in polymer or dimers of dye molecules ? appear as different color rather than expected ( methylene blue gives red or purple color) What are metachromatic substances?? Ionized So4, Po4 of cartilage Where you find it? ? Mast cell granules (heparin) & rER of Plasma cells Metachomasia

9. Special stain PAS positive substances ?Carbohydrate (glycogen) or carbohydrate rich molecules, Basement membrane, reticular fibers Periodic acid cleaves bond between carbon atoms ? form aldehyde group Aldehyde binds with Schiff to produce magenta or pink color PAS =Periodic Acid Schiff

10. Acid hydrolyses or cleaves proteins from deoxyribose of DNA ? leads to opening of sugar group & formation of aldehyde Schiff binds and gives magenta color to aldehyde Can be useful to quantify amount of DNA ( by using spectrophotmetry of Feulgen stained tissue) Feulgen stain for Nuclear Proteins

11. For the confirmation of specific substances Pretreatment of sections with specific enzymes Diastase/amylase ? for glycogen DNA ase ? for DNA Enzymatic digestion

12. Localization of enzymatic activity in tissues Best fixation – mild aldehyde ( formalin) Basis – localized reaction production of enzyme activity Used for acid & alkaline phosphatase, ATP ases AB (substrate) + T (trap) AT ( reaction product) + B (Hydrolyzed component of substrate) Enzyme Histochemistry

13. Antibody ( Immunoglobulin) conjugated with fluorescent dye( most common is Fluorescein) + Antigen ( foreign protein) Fluorescein ? absorbs UV light and emits green fluorescence ? can be seen under Fluorescent microscope (IF- Immuno Fluorescence) Example :- actin (Antigen) of Rat ? infected to Rabbit ? blood of Rabbit ( have poly - clonal antibodies for Rat’s actin/ anti rat actin antibodies) ? bind with Fluorescent dye Immuno Histo Chemistry (IHC)

14. Specific antigen (actin of rat) Monoclonal Antibodies

15. Diagnosis of tumors(tumor markers) & Infections( HIV, Infectious Mononucleosis) Classify sub – types (B -cell and T- cell lymphomas) Treatment – Anti-TNF-a antibodies in inflammatory disorders Clinical Significance of Monoclonal Antibodies

16. Immuno -fluorescence Direct (one step, less sensitive) & Indirect ( more sensitive, Expensive, labor intensive, can’t easily run in automated) methods Immunoperoxidase method Enzyme is used ( horse raddish peroxidase) to color colorless substrate into colored insoluble product Immunological Methods

17. Other Methods Hybridization: for localizing mRNA/DNA (NA) In Situ Hybridization: Binding ( Probe + NA) in cell/tissue FISH: If Fluorochrome is used in Hybridization technique Autoradiography: by tagging the precursor molecules (Amino acids) followed by synthesis of large molecules (NA) ? localize the particular tagged molecule

18. Microscopy Resolution/ Resolving power (RP): the distance by which two objects must be separated to be seen as two objects RP of Unaided Human retina : 0.2 mm Light Microscope (LM) : 0.2 ΅ Electro Microscope (EM) : 1.0 nm LM: we see only two dimensional pictures, orientation of cut gives different patterns Artifacts: error in preparation process

19. orientation of cut

20. Three dimensional picture

21. Types & Advantages of Microscopes 1. Phase contrast M: can see live (unstained) tissue Light passing thru denser tissue of higher refractory index ? out of phase from the rest ? look darker Uses : identify cells in tissue cultures Modification: Interference M: quantification of tissue masses helps in study of surface properties of cells

22. Types & Advantages of Microscopes 2. dark Field M: special condenser illuminates specimen with strong oblique light Uses: In auto radiography Study crystals in urine Study microbes- slender spirochetes ( *Treponema pallidum) 3. Fluorescent M: emits light in visible range when exposed to UV light Technique: filters are used between light source & specimen Naturally fluorescent substances: Vitamin “A”, Neuro- transmitters Uses Tracing pathways of nerve fibers, To detect growth markers of mineralized tissues

23. Types & Advantages of Microscopes 4. Confocal scanning M: Conjugate with focal point of lens Computer software reconstitutes the image from the data Major difference from LM: addition of detector aperture (pin hole) Uses: can see 3D pictures 5. ultra violet M: Depends on absorption of UVL by specimen Results are recorded photographically (can’t be seen directly – why?) Uses Study of nitrogen bases ( in NA) Study amount of DNA/RNA in cells? *Clinically helps in study of ploidy in tumors

24. Types & Advantages of Microscopes 6. Polarizing M: only difference is polarizer (polarizing filter) Birefringence: ability of crystalline or Para - crystalline material to rotate the phase of polarized light (double refraction) Skeletal muscle & Leydig cells *Amyloid protein: apple green ±Uric acid: negative ± ± Ca++ pyrophosphate

25. Types & Advantages of Microscopes 7. Electron M (EM): specimen is in vacuum Types: Transmission (TEM), scanning (SEM) Mechanism: similar to LM except that beam of electrons replace light source Recording: photoelectric plate or video detector Specimen preparation: Fixation: Glutaraldehyde (cross links with proteins), Osmium tetroxide (reacts with *phospholipids) makes cell/tissue electron dense for image enhancement Other steps are same as routine tissue processing except Plastic is used for embedding ± Diamond knives are used in microtome ( not metal knives) To study membranes – Freeze fracture technique {-160°C with glycerol (to prevent ice crystal formation)}

26. Types & Advantages of Microscopes 7. Scanning (SEM) It differs from TEM that electron beam passes across the surface of spectrum (not thru specimen as in TEM) Resembles Television Can see 3D pictures 8. Atomic Force M: most powerful tool to study surface topography Non – optical M: works like finger tip Has highest resolution power – 50 pm *Specimen need not be in vacuum

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