1. 1Slide Other Methods Northern blotting – analyzes mRNA expression in tissue In situ hybridization – Visualizing gene activity (mRNA) directly in fixed cells or tissues Western blotting – analyzes proteins expressed in tissue Immunohistochemistry (IHC) or Immunocytochemistry (ICC) – labels proteins in fixed cells or tissues Northern blotting – analyzes mRNA expression in tissue In situ hybridization – Visualizing gene activity (mRNA) directly in fixed cells or tissues Western blotting – analyzes proteins expressed in tissue Immunohistochemistry (IHC) or Immunocytochemistry (ICC) – labels proteins in fixed cells or tissues

2. 2Slide Northern Blotting Purpose: Can tell us if a particular gene is being expressed in a specific cell or tissue type or if there are changes in levels of expression ----------------------------------------------------------------------------------------------------------------------------------- General Procedure: Isolate mRNA from cells Separate mRNA with gel electrophoresis Blot (transfer) the mRNA to a membrane Label mRNA you are interested in (probe with a complementary sequence that binds your mRNA of interest) Purpose: Can tell us if a particular gene is being expressed in a specific cell or tissue type or if there are changes in levels of expression ----------------------------------------------------------------------------------------------------------------------------------- General Procedure: Isolate mRNA from cells Separate mRNA with gel electrophoresis Blot (transfer) the mRNA to a membrane Label mRNA you are interested in (probe with a complementary sequence that binds your mRNA of interest)

3. 3Slide In situ Hybridization Purpose: Allows us to visualize gene activity directly in tissues or cells ----------------------------------------------------------------------------------------------------------------------------------- General Procedure: Tissues may be cut into thin sections or entire embryo may be adhered to a microscope slide mRNA probes are labeled with a dye or enzyme that makes a colored precipitate Tissues are imaged with a microscope Purpose: Allows us to visualize gene activity directly in tissues or cells ----------------------------------------------------------------------------------------------------------------------------------- General Procedure: Tissues may be cut into thin sections or entire embryo may be adhered to a microscope slide mRNA probes are labeled with a dye or enzyme that makes a colored precipitate Tissues are imaged with a microscope

4. 4Slide Western Blotting Purpose: Detects levels of proteins being produced in a particular cell or tissue ----------------------------------------------------------------------------------------------------------------------------------- General Procedure: Isolate proteins Separate proteins with gel electrophoresis Blot (transfer) the proteins to a membrane Label protein you are interested in (antibody that binds specifically to your protein) Bands can tell if protein is present and if there are different molecular forms of it Purpose: Detects levels of proteins being produced in a particular cell or tissue ----------------------------------------------------------------------------------------------------------------------------------- General Procedure: Isolate proteins Separate proteins with gel electrophoresis Blot (transfer) the proteins to a membrane Label protein you are interested in (antibody that binds specifically to your protein) Bands can tell if protein is present and if there are different molecular forms of it

5. 5Slide Example of Western Blotting EXNE Turkey NBTurkey BR FemaleMale EXNEEXNEEXNE Zebra finch anterior pituitaries Breeding Not Breeding Prolactin Isoforms in the Anterior Pituitary of Zebra Finches The lane on the extreme left is the ladder (mol wt marker). Tissue extracts in the remaining lanes are labeled with a prolactin antibody. Some birds demonstrate a second, slightly heavier band of prolactin. Because it is heavier, it migrates more slowly, and shows prolactin exists in two forms in these birds; the heavier prolactin has been modified by adding a sugar group.

6. 6Slide Microscopy The idea behind any type of microscopy is to see inside the tissue and/or cell. What we see and how well we see it, depend upon: 1. Type of microscope 2. Tissue preparation 3. Tissue staining The idea behind any type of microscopy is to see inside the tissue and/or cell. What we see and how well we see it, depend upon: 1. Type of microscope 2. Tissue preparation 3. Tissue staining

7. 7Slide Types of Microscopes Light Microscope: Resolution of about 0.2  m Good for routine staining Fluorescent microscope: Fluorescent dyes are excited by specific wavelength of light and re-emit it at higher wavelengths (lower energy). (ex: FITC – a very common fluorescent dye – is excited at 488, emits at 525nm - looks green) Filters eliminate stray light, so the image is sharper Light Microscope: Resolution of about 0.2  m Good for routine staining Fluorescent microscope: Fluorescent dyes are excited by specific wavelength of light and re-emit it at higher wavelengths (lower energy). (ex: FITC – a very common fluorescent dye – is excited at 488, emits at 525nm - looks green) Filters eliminate stray light, so the image is sharper

8. 8Slide Confocal Microscope Uses a laser to excite fluorescent dye in tissue or cell one spot at a time Precisely positioned pinholes allow only in- focus light to pass through Images in XYZ planes Computer takes all images and creates a 3D reconstruction

9. 9Slide Confocal microscope: 0.2-0.5  m resolution 3D images can be obtained http://www.jneurosci.org/content/vol26/issue 46/images/data/11870/DC1/Fig._12C- PBC-3D_animation- Ruangkittisakul_et_al.mov Ion movements can be recorded ]http://www.jneurosci.org/content/vol26/issue 46/images/data/11870/DC1/Fig._12A- PBC_calcium_oscillations- Ruangkittisakul_et_al.movhttp://www.jneurosci.org/content/vol26/issue 46/images/data/11870/DC1/Fig._12A- PBC_calcium_oscillations- Ruangkittisakul_et_al.mov Confocal microscope: 0.2-0.5  m resolution 3D images can be obtained http://www.jneurosci.org/content/vol26/issue 46/images/data/11870/DC1/Fig._12C- PBC-3D_animation- Ruangkittisakul_et_al.mov Ion movements can be recorded ]http://www.jneurosci.org/content/vol26/issue 46/images/data/11870/DC1/Fig._12A- PBC_calcium_oscillations- Ruangkittisakul_et_al.movhttp://www.jneurosci.org/content/vol26/issue 46/images/data/11870/DC1/Fig._12A- PBC_calcium_oscillations- Ruangkittisakul_et_al.mov Anterior pituitary of a zebra finch. Cells secreting prolactin appear red, while those secreting growth hormone appear green. An individual cell in the lower right corner has both hormones and appears yellow. The dark “hole” in the middle of the cell is its nucleus. From: Christensen, 2007 Confocal Microscope

10. 10Slide Tissue Preparation Fresh tissue is thick, mushy, and opaque. Also has active enzymes and bacteria that promote degradation. 3 basic steps: 1. Fixation – stabilizes structure 2. Embedding & sectioning – makes it hard so we can cut thin slices 3. Staining – provides contrast so we can see it easier Fresh tissue is thick, mushy, and opaque. Also has active enzymes and bacteria that promote degradation. 3 basic steps: 1. Fixation – stabilizes structure 2. Embedding & sectioning – makes it hard so we can cut thin slices 3. Staining – provides contrast so we can see it easier

11. 11Slide Histological Techniques Fixation: stabilizes structure stops enzymes prevents bacterial degradation (putrefaction) The most common fixatives cross-link tissue proteins. An example is formaldehyde. Fixation: stabilizes structure stops enzymes prevents bacterial degradation (putrefaction) The most common fixatives cross-link tissue proteins. An example is formaldehyde.

12. 12Slide Embedding & Sectioning Embedding: Infiltrate tissue with harder substance that is more easily cut into thin sections 1. Resin – plastic, used for very thin sections, 0.5-1µm 2. Paraffin – used for sections >5µm 3. Freezing – used for sections 20-100µm Embedding: Infiltrate tissue with harder substance that is more easily cut into thin sections 1. Resin – plastic, used for very thin sections, 0.5-1µm 2. Paraffin – used for sections >5µm 3. Freezing – used for sections 20-100µm

13. 13Slide Sectioning Paraffin (frozen similar) 1. Sectioned with a rotary microtome 2. Metal or glass knife or razor blade 3. Sections to 1-2 µm thick Paraffin (frozen similar) 1. Sectioned with a rotary microtome 2. Metal or glass knife or razor blade 3. Sections to 1-2 µm thick

14. 14Slide Staining Staining - introduces contrast to tissue which is otherwise essentially transparent Common stains: Hematoxylin (looks blue-purple) & eosin (looks pink-red) Usually hematoxylin and eosin are used together (H&E) Staining - introduces contrast to tissue which is otherwise essentially transparent Common stains: Hematoxylin (looks blue-purple) & eosin (looks pink-red) Usually hematoxylin and eosin are used together (H&E)

15. 15Slide HistochemistryHistochemistry Can stain DNA & visualize replicating cells

16. 16SlideDNADNA BRd-U - bromodeoxyuridine - analog of thymidine, incorporated when DNA replicates a. Antibody stains labeled cells b. Can follow time sequence of cell division BRd-U - bromodeoxyuridine - analog of thymidine, incorporated when DNA replicates a. Antibody stains labeled cells b. Can follow time sequence of cell division

17. 17Slide Immunohistochemistry (IHC) Advantages 1. High specificity for molecular species 2. Can be used for light, confocal, or EM Advantages 1. High specificity for molecular species 2. Can be used for light, confocal, or EM Disadvantages 1. 1.Time consuming & expensive 2. Fixation can interfere with Ab binding 3. Reproducibility - false positives - cross reactivity 4. Difficult to get Abs to small molecules Uses antibodies to detect specific molecules in tissue

18. 18Slide Immuno methods 1. Direct method - label (dye) is on the antibody

19. 19Slide 2. Indirect method - label is on a secondary antibody, amplifies label. Primary antibody binds to molecule of interest Second antibody (labeled with dye) against species in which primary was raised - eg, goat anti-rabbit antibody 2. Indirect method - label is on a secondary antibody, amplifies label. Primary antibody binds to molecule of interest Second antibody (labeled with dye) against species in which primary was raised - eg, goat anti-rabbit antibody Immuno methods

20. 20Slide Labels Fluorescent dyes - FITC, rhodamine, Cy3, Cy5 - different excitation and emissionspectra allows double labeling Enzymes (horse-radish peroxidase (HRP) or alkaline phosphatase) give colored precipitate Fluorescent dyes - FITC, rhodamine, Cy3, Cy5 - different excitation and emissionspectra allows double labeling Enzymes (horse-radish peroxidase (HRP) or alkaline phosphatase) give colored precipitate