Module IB. Histochemistry. Martin Špaček, MD. (

Transcription

1 Module IB Histochemistry Martin Špaček, MD (

2 What is histochemistry? It is a histological technique used for studying chemistry of tissues and cells Histochemistry Enzyme histochemistry Immunocytochemistry In situ hybridization

3 Histochemistry Examples of Histochemical Methods: Ions Iron (ferric ions Fe 3+ ) Perls s reaction: sections of tissues are incubated in a mixture of potassium ferrocyanide and hydrochloric acid result: insoluble dark blue precipitate of ferric ferrocyanide Diagnostic application: patients with diseases that store iron (eg. hemochromatosis)

4 Spleen Perls (16)

5 Histochemistry Examples of Histochemical Methods: Lipids stained with dyes soluble in the lipids eg. Sudan IV, Sudan black, Oil red, Nile blue fresh frozen (cryostat) sections are used for the most authentic picture of tissue lipids

6 Histochemistry Examples of Histochemical Methods: Nucleic Acids DNA Feulgen s reaction: hydrolysis of DNA by hydrochloric acid this process leads to the formation of aldehyde groups free aldehyde groups react with the Schiff reagent result: insoluble red substance

7 Histochemistry Examples of Histochemical Methods: Nucleic Acids RNA RNA-rich organelles are stained with basic dyes i.e.: toluidine blue, methylene blue Since the RNA is not the only basophilic substance in the tissue it is necessary to incubate a control slide with ribonuclease

8 Kidney methylene blue (20)

9 Histochemistry Examples of Histochemical Methods: Saccharides Saccharides can be detect by PAS reaction (periodic acid-schiff) it is based on oxidative action of periodic acid (HIO 4 ) aldehyde groups these aldehyde groups react with Schiff s reagent (as in Feulgen s reaction) a new compound with a purple colour (PAS-positive substances)

10 Histochemistry Examples of Histochemical Methods: Saccharides PAS-positive substances are: polysaccharides (glycogen) glycosaminoglycans /mucopolysaccharides/ (hyaluronic acid, chondroitin sulphate) proteoglycans glycoproteins (thyreoglobulin, collagen) glycolipids (lipofuscin) Clinical application: biopsies of tissues from patients with diseases that store glycogen (glycogenosis), glycosaminoglycans (mucopolysaccharidosis)...

11 Spleen PAS (17)

12 Colon PAS (19)

13 Colon PAS (19)

14 Enzyme Histochemistry Enzymes are the catalysts of most of biochemical reactions Frozen tissues are used Principle: 1 st reaction (histochemical): enzyme + substrate product 2 nd reaction: demonstration of the product

15 Peroxidase Enzyme Histochemistry Examples sections are incubated in a solution containing hydrogen peroxide and DAB (diaminoazobenzidine) DAB is oxidized in the presence of peroxidase result: insoluble, black, electrondense precipitate Clinical application: diagnosis of leukemias (detection of the peroxidase activity in blood cells) Peroxidase is used also as a label in immunocytochemistry and in ISH

16 Enzyme histochemistry Alkaline phosphatase in the proximal tubules of the kidney

17 Enzyme histochemistry Demonstration of aminopeptidase M in the proximal tubules of the kidney

18 Immunocytochemistry Is a technique for identifying cellular or tissue constituents (antigens) by means of antigen-antibody interactions Antigens: proteins, glycoproteins, proteoglycans Antibodies: serum proteins known as immunoglobulins formed in the humoral immune system by plasma cells there are five types of antibody found in the blood IgA, IgD, IgE, IgG and IgM. IgG is the commonest and the most frequently used antibody for immunohistochemistry

19 Antibodies An antibody molecule is composed of four polypeptide chains, two identical light chains and two identical heavy chains

20 Antigenic Determinants (Epitopes) The specific site of an antigen that binds to an antibody is called an antigenic determinant or epitope Most antigens have a variety of epitopes that generate a number of different antibodies that are called polyclonal A single immune response to an antigen is termed monoclonal

21 Monoclonal antibodies Monoclonal antibodies are cloned antibodies that all respond to exactly the same epitope of the same antigen they are exceptionally specific Activated B cells (plasma cells) contain a mixture of antibodies directed against different epitopes Activated B cells have a limited lifetime when grown in culture How can we select a single B cell clone (monoclonal) and propagate the cell line?

22 Monoclonal antibodies Antibodies can be raised in mice by injection of an antigen. Repeated injections create a pool of activated B cells.

23 Monoclonal antibodies B lymphocytes can mutate into tumor cells that result in a type of cancer termed myeloma. Myeloma cells become immortal and will grow indefinitely in culture. Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinitely in culture

24 Methods of Labelling Antibodies Enzyme labels (peroxidase) Colloidal metal labels (EM) Fluorescent labels Avidin-biotin techniques

25 Fluorescent Compounds Fluochromes are substances which can absorb radiation in the form of ultraviolet or visible light This absorbed radiation causes an excited state followed by emission of radiation of a different wavelength The fluorescent substances then appear as shiny particles on a dark background Fluorochromes: FITC (fluorescein isothiocyanate), rhodamine, DAPI, etc.

26 Fluorescent labels The chromatine is stained with a blue fluorescent probe DAPI

27 Fluorescent labels FITC Appearance Na+/K+ ATPase in basolateral labyrint of proximal and distal tubule of kidney

28 Avidin-biotin Techniques These methods rely on the affinity of the glycoprotein avidin for biotin Biotin, a low molecular weight vitamin, is easily conjugated to antibodies and enzyme markers Up to 150 biotin molecules can be attached to one antibody molecule increased sensitivity

29 Skin / keratine ABC (22)

30 Skin / Melanocytes and Langerhans cells

31 Anti - S antibody, eccrine sweat glands of the skin

32 Pancreas / insulin ABC (21)

33 Pancreas / somatostatin in the D cells of the islets of Langerhans

34 Antibody labels neurofilaments of neurons in the cortex of brain

35 Antibody labels neurofilaments in Purkinje cells and in axons of basketlike cells

36 Cerebrum / astrocytes ABC (96)

37 Methods of Localizing Antigens Direct method primary antibody is conjugated directly to the label Indirect method (more sensitive) first step: nonlabeled anti-x antibody is bound to the antigen second: the labeled antiantibody binds to the anti-x antibody

38 In Situ Hybridization Techniques Allow the demonstration of specific nucleic acid sequences in their cellular environment Are based on hybridization of a nucleic acid probe (small lengths of nucleic acid of known base sequence) to the target Applications: detection of abnormal genes identification of viral infection, etc.

39 In Situ Hybridization Techniques Probes labelling: radioisotopes coupling of the nucleotides with biotin fluorescent labels (FISH)

40 Fluorescence in Situ Hybridization (FISH)

41 Fluorescence in Situ Hybridization (FISH)

42 Immunohistochemistry + ISH Human Papillomavirus DNA demonstrated by In Situ Hybridisation (pink) in epithelial cells identified by indirect immunofluorescence using antibody against cytokeratin (green)