1. Immunohistochemistry Because I couldn’t think of a witty title
Karen Eskow Jaunarajs

2. A Haiku By Cara Martin Hueston
DAB turns brown,
mapping protein in a cell.
where is the TH?
A technique which enables the mapping of a protein in relation to the cell and tissue

3. Basic Procedure Perfusion and Tissue Sectioning
PBS
PFA

4. Basic Procedure Protein Labeling
Washing with PBS
0.3% Hydrogen Peroxide (20-30 min)
Wash with PBS
Blocking buffer (1 h)
Primary antibody (2-48 h)
Secondary antibody (1 h)
Avidin-biotin complex (ABC; 1 h)
DAB (20 s-15 min)
Mount on slides

5. Quantification of Ir+ Cells Profile Counting, Optical Density, or Stereology
Count all cells in a structure of interest
Usually necessary when few ir+ cells (<100)
Optical Density
Use an optical scanner to quantify darkness of staining in a certain area of interest
Useful when cells are too close together to count, there are multiple grains of ir+ protein within one cell, or counting neuronal fibers

6. Quantification of Ir+ Cells Profile Counting, Optical Density, or Stereology
Unbiased Stereology
Counting a consistently random portion of a structure and applying equations which estimate the total number of ir+ cells within that 3D structure
Area of interest
Counting frame
Volume measurement
Requires a number of tissue sections (at least 5/structure/animal) and >100 total ir+ cells/structure

7. Application in the Bishop Lab
Tyrosine Hydroxylase
Verification of DA lesion
Effects on locus coeruleus (NE neurons)
Serotonin (5-HT)
Effects of L-DOPA on 5-HT
ERK1/2; MAPK
Effects of L-DOPA or DA agonists on neuronal signaling
Effects of 5-HT1A agonists on neuronal signaling
Cfos
Effects of L-DOPA/5-HT1A agonists on cellular activation

8. Other Cool Variations
Immunofluorescence
Double Labeling

9. A Limerick by Cara Martin Hueston (who is not from Limerick)
If beautifully stained brains make you gloat Make sure you use donkey-anti goat! IHC may be the answer, But since DAB gives you cancer, You should also wear a snazzy lab coat.