Naslov
Problems in immunohistochemical analysis of lipid-anchored molecules

Autori
Heffer-Lauc, Marija ; Viljetić, Barbara ; Vajn, Katarina ; Lauc, Gordan

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, ostalo

Skup
2nd Croatian Congress on Microscopy

Mjesto i datum
Topusko, Hrvatska, 18.05.2006. - 21.05.2006

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
lipid-anchored molecules; detergents; immunohistochemistry

Sažetak
Several types of molecules are bound to the membrane via lipid anchor that is inserted into one of the phospholipid leaflets in the membrane. Glycolipids are always situated in the outer membrane leaflet, while proteins can be anchored either in the outer leaflet (via GPI– anchor), or in the inner leaflet (via prenil, myristil or farnezil anchors). Immunochemical detection of these molecules in the brain is somewhat complex because of extensive myelin sheets that block access of antibodies to cell membranes and require pre-treatment with detergents. Using mice deficient for complex gangliosides or GPI-anchored protein Thy-1 we recently demonstrated that pretreatment with Triton X-100 in the concentration range of 0.1% to 1% for 2 h causes artifacts due to redistribution and shedding of glycolipids and GPI-anchored proteins. Since membranes have to be made porous to enable to enable detection of axonal epitopes, we tested several detergents hoping to find one that makes pores in membrane, but does not result in significant redistribution of lipid-anchored molecules. In addition to detergent, we also tested repeated freezing and thawing and electroporation as a way to permeabilize membranes. Co-incubation of knockout and wild-type mice was used to examine redistribution, while permeabilization of membranes was tested by immunohistochemical analysis of MAG (myelin associated glycoprotein). Our results clearly demonstrate that routine immunohistochemical protocol needs to be altered when used for the analysis of lipid-anchored molecules and that additional evidence might be needed to confirm their real physiological localization.

Izvorni jezik
Engleski

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