Detecting tagged Hedgehog with intracellular and extracellular immunocytochemistry for functional analysis.
In this chapter, we explain different strategies to analyze the extracellular Hedgehog (Hh) morphogen distribution and Hh intracellular trafficking by immunohistochemistry techniques. For this purpose, it has been very useful to have a transgenic fly line that expresses a Hh-green fluorescent protein (GFP) fusion protein. These flies can be used to study the way Hh spreads through the anterior compartment where it signals, and analyze in detail how Hh is internalized by its receptor Patched. In addition, this Hh-GFP fusion made without lipid modifications (cholesterol or palmitic acid) can be used to investigate the function of these lipids on Hh in terms of spreading, internalization, and signaling abilities.
Callejo
Ainhoa
A
Quijada
Luis
L
Guerrero
Isabel
I
D000818:Animals; D030801:Animals, Genetically Modified; D004330:Drosophila; D004705:Endocytosis; D005110:Extracellular Space / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D053823:Hedgehog Proteins / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods*; D042541:Intracellular Space / Q000378:metabolism*; D018613:Microscopy, Confocal; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility; D013194:Staining and Labeling; D014921:Wings, Animal / Q000033:anatomy & histology / Q000166:cytology
D053823:Hedgehog Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins