1. ANNOUNCEMENTS Help / Review Session Fridays 12:20 Morrill 349 Lab Today: Please read the material for Lab 1! TODAY: Microscopy

2. Summary of Staining Approaches to Date 1) Charged dyes stain molecules of opposite charge (basic dye-> negative molecules) 2) Stains/dyes react with specific class of molecules (sugars, lipid) 3) Enzymatic digestion of stained molecules of interest (amylase-> glycogen)

3. Staining Techniques Localization (staining) of an Enzyme AB + T AT + B ENZYME generate visible product provide substrate

4. Staining Techniques AB + T AT + B Acetylcholinesterase- neuromuscular junction ACETYL CHOLINESTERASE Other stains for ATPases, alkaline phosphatases, and others

5. IMMUNOCYTOCHEMISTRY Use of antibodies to detect the presence of specific molecules (antigens) in a tissue. Antibody binds to an antigen. Specific binding site on antigen-->Epitope ANTIGEN ANTIBODY

6. IMMUNOCYTOCHEMISTRY Direct Immunocytochemistry: a visible marker is directly attached to an antibody that binds an antigen. The antibody is conjugated to a visible marker. Possible Markers ( ): Fluorochrome Enzyme (HRP) Electron dense molecule ferritin, gold

7. Direct Immunocytochemistry Fix the tissue Rinse with saline solution Incubate with conjugated antibody Rinse Mount on slide and view under microscope

8. DIRECT IMMUNOCYTOCHEMISTRY ADVANTAGES Specificity Less background staining DISADVANTAGES Low sensitivity if the antigen is present in the tissue in low concentrations. Need to directly conjugate marker to antibody.

9. INDIRECT IMMUNOCYTOCHEMISTRY –Primary unlabeled antibody binds to the antigen. –A Secondary labeled antibody binds to the primary antibody.

10. INDIRECT IMMUNOCYTOCHEMISTRY –Primary antibody binds to the antigen. –Secondary antibody (conjugated to a visible marker) binds to the primary antibody.

11. INDIRECT IMMUNOCYTOCHEMISTRY Fix the tissue Rinse Incubate with unlabeled primary antibody Rinse Incubate with labeled secondary antibody Rinse Mount the tissue and view under the microscope

12. INDIRECT IMMUNOCYTOCHEMISTRY ADVANTAGES Amplification of the signal Can use labeled secondary with different primary antibodies DISADVANTAGES Nonspecific background may increase Takes longer to do Needs more reagents

13. LIMITATIONS OF IMMUNOCYTOCHEMISTRY Cross-reactivity Sensitivity Antigenicity

14. LIMITATIONS OF IMMUNOCYTOCHEMISTRY Cross-reactivity Sensitivity Antigenicity -Frozen sections

15. Types of antibodies used in immunocytochemistry

16. Polyclonal antibodies -A pool of different antibodies recognize different epitopes on the same antigen

17. Types of antibodies used in immunocytochemistry Monoclonal antibodies -A single antibody recognizes only 1 epitope

18. Types of antibodies used in immunocytochemistry Polyclonal antibodies ADVANTAGES: recognize more epitopes in tissue DISADVANTAGES: less specificity Monoclonal antibodies ADVANTAGES: more specific DISADVANTAGES: reduced signal possible

19. Antibodies (immunoglobulins) of specific species are used as antigens to generate secondary antibodies. ANTIGEN--> mouse antibody Rabbit anti-mouse IgG Goat anti-mouse IgG Donkey anti-rabbit IgM

20. QUESTION: Dr. Reist is studying the distribution of two proteins, FasII and spectrin in neurons. She would like to label both molecules in the same sample using double- labeling immunocytochemistry. She has these antibodies: Primary antibodies:Secondary antibodies: rabbit anti-FasII mouse anti-rabbit-FITC(fluorescein) mouse anti-FasII donkey anti-rabbit-FITC goat anti-FasII rat anti-mouse-Rh (rhodamine) rat anti-spectrin goat anti-mouse-Rh rabbit anti-spectrinrabbit anti-Goat-Rh donkey anti-spectrin What primary and secondary antibodies will successfully distinguish the distribution of FasII and spectrin in the same preparation?

21. Primary antibodies:Secondary antibodies: rabbit anti-FasII mouse anti-rabbit-FITC(fluorescein) mouse anti-FasII donkey anti-rabbit-FITC goat anti-FasII rat anti-mouse-Rh (rhodamine) rat anti-spectrin goat anti-mouse-Rh rabbit anti-spectrinrabbit anti-Goat-Rh donkey anti-spectrin

22. AUTORADIOGRAPHY Tissue incubation with radiolabeled molecule Fixation and mounting Prep covered with photographic emulsion Exposure and development

25. In situ hybridization Labeled DNA or RNA probe Why?

26. In situ hybridization Labeled DNA or RNA probe Radioactive tag Digoxigenin Incubation with tissue Autoradiography or Immunocytochemistry

27. www-bioc.rice.edu/bios576/immuno/immuno.html

29. Whole mount in situ hybridization views on E10.5 mouse embryos with Phox2a (A), En1 (B), Uncx4.1 (C) and Lmx1b (D) RNA-probes. Juha PartanenInstitute of Biotechnology, P.O.Box 56, FI-00014 Univ. of Helsinki

30. FOUR TISSUES - Epithelial Tissue - Connective Tissue - Nervous Tissue - Muscle Tissue

31. EPITHELIAL TISSUE -covers surfaces -lines cavities -forms glands

32. EPITHELIAL TISSUE Functions: 1) protect against abrasion 2) physical barrier 3) transport 4) excretion/secretion 5) sensation

33. CLASSIFICATION -Exhibit Polarity Free surface-lumen Apical Basal Lateral -Close cell apposition adhere to one another specialized cell junctions -Attached to a Basement Membrane=Basal Lamina

34. -Number of cell layers -Cell shape of surface cells -Specialization of apical surface CLASSIFICATION of EPITHELIA

35. -Number of cell layers Simple Stratified Pseudostratified CLASSIFICATION

36. Squamous: flat Epithelial Cell Shape

37. Squamous: flat width > height Simple squamous: Single cell layer Epithelial Cell Shape Blood vessels- endothelium Body cavity- mesothelium Kidney Lung

38. Squamous: flat Stratified squamous: >1 cell layers [Basal cells: stem cells for upper layers.] CLASSIFICATION Skin (epidermis) Mouth & Esophagus Vagina Anal canal

39. Keratinized Stratified Squamous Epithelia Skin Anal Canal Apical surface has a hardened layer of dead cells rich in keratin intermediate filaments. provides protection against abrasion and dessication (drying).

40. Cuboidal: height of cells = width Simple: (eg. thyroid, ovary, kidney tubules) Stratified: (sweat glands) CLASSIFICATION

41. Columnar: height of cells > width Simple: can be ciliated intestine gall bladder fallopian tubes CLASSIFICATION

42. Columnar: Stratified: (ducts of exocrine glands) CLASSIFICATION

43. Columnar: Pseudostratified: single cell layer that appears stratified; each cell in contact with basement membrane; frequently ciliated Trachea Urethra CLASSIFICATION

44. Transitional Epithelium : stratified, domed superficial cells, allows for extension; bladder