Specialized Prep Techniques

1. Specialized Prep Techniques Labels: • To identify the composition of the structure/organelle and/or phase of cycle. • Identification of questionable structures. • Follow cytochemical pathway through a time course. • Localize genetic products, introduced compounds. Cryo-fixation: • Structures are ephemeral or events are rapid. • Structures are fixative sensitive. • Removal of water changes topography/morphology

2. Low Temperature Techniques Freeze Fracture – Sample is rapidly frozen, fractured and replica is produced. Viewed with TEM. Cryo-Fixation – Can use a variety of methods to fix tissue prior to substitution (removal of water). Cryo-Microtomy– Used in conjunction with cryo-fixation to maintain frozen state of the sample. Sections are kept frozen for viewing or processed to dryness for conventional viewing. Freeze Drying – Easy and common technique for SEM.

3. Labeling Autoradiography – Introducing an isotope, then causing a reaction which deposits an electron dense material at radioactive sites. Lectin – Lectins are small molecular weight compounds that have variable affinities for sugars. Usually bound directly to label. Cytochemistry – Chemical reactions that require precipitation of colored reaction product, heavy metal or electron dense material for visualization. Immunolabeling – Polyclonal or monoclonal antibodies used to label components. Usually used with fluorescent dye, colloidal gold and sometimes silver enhancement. In Situ Hybridization – Using nucleic acid hybridization techniques with biotinylated nucleotides.

4. Immune Responses 1. Humoral: B lymphocytes produce antibodies recognizing an antigen from foreign substance. Antibodies are then secreted into blood stream.

5. Immune Responses 1. Humoral: B lymphocytes produce antibodies recognizing an antigen from foreign substance. Antibodies are then secreted into blood stream. 2. Cell-mediated: Mature T lymphocytes - antigen responding, response control, and response mediating cells

6. Immunoglobins (Ig) IgG

7. Immunoglobins (Ig) IgG IgA

8. Immunoglobins (Ig) IgG IgA IgM

9. Glossary Antibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte).

10. Glossary Antibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte). Antigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.g hapten).

11. Glossary Antibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte). Antigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.g hapten). Immunogen : Any substance to which an animal responds by making antibodies. All immunogens are antigens.

12. Glossary Antibody (anti-foreign body) is a protein produced by a white cell (B lymphocyte). Antigen (antibody generating substance) is any agent, such as a chemical or microorganism that is recognized by the antibody. Not all antigens are immunogens (e.g hapten). Immunogen : Any substance to which an animal responds by making antibodies. All immunogens are antigens. Antigen binding site - relatively small region of an antibody that binds to the antigen.

13. Epitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody.

14. Epitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody. Hapten - low molecular weight compounds (such as plant hormones) that typically do not elicit a spontaneous immune response but can be recognized by antibodies. Typically attached to an immunogen.

15. Epitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody. Hapten - low molecular weight compounds (such as plant hormones) that typically do not elicit a spontaneous immune response but can be recognized by antibodies. Typically attached to an immunogen. Hybridoma - fusion product between B cell and myeloma cell (“immortal cell”).

16. Epitope (antigenic determinant) - is that part of an antigen that is recognized by a single antibody. Hapten - low molecular weight compounds (such as plant hormones) that typically do not elicit a spontaneous immune response but can be recognized by antibodies. Typically attached to an immunogen. Hybridoma - fusion product between B cell and myeloma cell (“immortal cell”). HAT selection - culture media that contains hypoxanthine, aminopterin and thymadine. A selective media that only allows hybridomas to grow.

17. Terms used in Immunolabeling Primary antibody: An antibody that is specific to the antigen of the sample to be localized. Can be conjugated to a signal (gold, fluorochrome or enzyme).

18. Terms used in Immunolabeling Primary antibody: An antibody that is specific to the antigen of the sample to be localized. Can be conjugated to a signal (gold, fluorochrome or enzyme). Secondary antibody: An antibody that recognizes a primary antibody. Usually always is conjugated to signal.

19. Terms used in Immunolabeling Primary antibody: An antibody that is specific to the antigen of the sample to be localized. Can be conjugated to a signal (gold, fluorochrome or enzyme). Secondary antibody: An antibody that recognizes a primary antibody. Usually always is conjugated to signal. Diluent: Physiologic buffer and non-specific protein (e.g. albumin or non-fat milk) used in diluting the antibodies. Sometimes detergent added to decrease surface tension of sections.

20. Block: Physiologic buffer, high salt, and non-specific protein. The protein adheres to any “sticky” sites that might allow non-specific binding of antibodies.

21. Block: Physiologic buffer, high salt, and non-specific protein. The protein adheres to any “sticky” sites that might allow non-specific binding of antibodies. Etching: treating resin sections with HCl or sodium borohydride to reduce steric hindrance or expose hidden antigenic sites.

22. Antibody Production Polyclonal: Antibodies are collected from sera of exposed animal, - or - a combination of monoclonal colonies is combined.

23. Antibody Production Polyclonal: Antibodies are collected from sera of exposed animal, - or - a combination of monoclonal colonies is combined. Can be any animal: Rabbit, Goat, Horse, Rat, Sheep, etc…

24. Antibody Production Polyclonal: Antibodies are collected from sera of exposed animal, - or - a combination of monoclonal colonies is combined. Can be any animal: Rabbit, Goat, Horse, Rat, Sheep, etc… Suite of antibodies recognizing multiple antigenic sites of injected biochemical.

25. Monoclonal: Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media.

26. Monoclonal: Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media. Typically BALBc mice Sometimes Rat (ascites fluid).

27. Monoclonal: Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media. Typically BALBc mice Sometimes Rat (ascitesfluid). Antibodies recognize one antigenic binding site of the antigen.

29. Determining which B cell to clone ELISA or Immuno-fluorescence

30. Generic light-level Immunolabeling Protocol Fixation: Formaldehyde (1-4% in physiological buffer) Incubation in 10% Triton X-100 for 10 minutes (may require additional 10 minutes in Methanol) Rinsing/rehydration in buffer Immunolabel Mount in glycerol-based medium or permanent mount. Usually contains anti-fade (anti-quench) agent.

31. Generic TEM Immunolabeling Protocol Fixation: 1% or less glutaraldehyde only – may include paraformaldehyde Dehydration Embedding in methacrylate resin (e.g. LR White, Lowicryl, or Quetol) Section Immunolabel Optional - Post-stain

32. Controls Adsorption - primary antibody exposed to excess of antigen to remove any labeling. Label without antibody - no antibody, shows labeling not due to reaction with label Omission of primary antibody - the secondary or tertiary antibodies should not recognize the tissue. Pre-immune sera - collected from animals that have not produced antibodies, or use “normal serum” from non-immunized animals of same species

33. Immunolabeling Sections - Float grids on blocking solution - Incubate in primary antibody - Wash thoroughly with buffer to remove unbound antibody

34. Incubate with secondary antibody or protein A/G Wash again to remove unbound secondary Last wash should be water.

35. Double Labeling Incubate sections with second primary antibody of interest

36. Incubate with secondary – different fluorochrome, or different size gold Note steric hinderence at asterisk

37. 10 nm and 5 nm gold

39. Microtubules labeled with anti-beta tubulin

40. How are the antibodies coupled to the gold?The proteins are attached to the gold particles by: • a). Charge attraction (Lysine) • b). Hydrophobic attraction (Tryptophan) and • c). Sulfur binding (Cysteine and Methionine) Strongest binding suggested to be the sulfur "hinge" joining the the two Fc regions

41. Using Protein A or Protein G instead of secondary antibody

42. Ferritin enhanced labeling Silver enhanced

43. Freeze fracture immunolabeled

44. Freeze fracture immunolabeled Negative stain Immunolabeled

45. Etching to expose antigenic sites

46. Arabidopsis root with anti-carbohydrate Abs

47. Lectins The term “lectin” is a general term that encompasses several families of proteins of non-immune origin that bind glycoconjugates that may or may not have a known function. Also known as agglutinins since original discoveries used agglutination of red blood cells (recognition of surface sugars) as a criteria. Many have secondary or even tertiary affinities to other sugars - rigorous controls required.

48. 1. A lectin molecule contains at least two sugar-binding sites; sugar-binding proteins with a single site will not agglutinate or precipitate structures that contain sugar residues, so are not classified as lectins. 2. The specificity of a lectin is usually defined by the monosaccharides or oligosaccharides that are best at inhibiting the agglutination or precipitation the lectin causes. 3. Lectins occur in many types of organisms; they may be soluble or membrane-bound; they may be glycoproteins. 4. Sugar-specific enzymes, transport proteins and toxins may qualify as lectins if they have, multiple-sugar binding sites. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), and Nomenclature Commission of IUB (NC-IUB)

49. General Classes of Lectins Animal 1) Galectins share galactose-specificity. 2) Ca-dependent (C-type) animal lectins form an extremely large family, composed of members having diverse structures and functions. Selectins are a subfamily that have a specific function in leukocyte adhesion to endothelial cells through sialyl-LewisX recognition. Collectins have a unique structure consisting of a C-type lectin domain and a collagen-like domain. They are supposed to be involved in innate immunity.

50. 3) Invertebrates (such as snails - e.g. Helix pomatia) are known to contain various lectins in their body fluids, possibly as body-protection factors. E.g. lectins from an echinoderm were found to show hemolytic activity. 4) Annexins have affinity to lipids with some having binding activity to glycosaminoglycans.