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

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

Basic Procedure Perfusion and Tissue Sectioning PBS PFA

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

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

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

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

Other Cool Variations Immunofluorescence Double Labeling

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.