Fluorescence

FIXIERUNG:variable–should be suited for antigen characteristics (Localisation, Structure, Conformation) and primary antibodies;Usually protocol can be interrupted AFTER fixation is completed – cells on ice (in PBS [Phosphate-buffered-saline –50mM Phosphatpuffer, pH 7.2, 150 mM NaCl]).MetOH:not for integral membrane proteins; it‘s mere protein-Precipitation!! Should becool to cold at –20oC;minimizes lipid extraction and improves fixation quality; isa quick procedure and should not last too long – 3 to 5 min to ensure structuralintegrity. Often in combination (2:1 oder 1:1) with Acetone; Usually cells arealready permeabilized.!! Perfect for cytoskeletal structures !!Formaldehyde/Para-Formaldehyde:some antibodies don‘t like it; Concentration-range: 1-4 % (para-f. Sometimes upto 8 %); Room-Temperature! Over-fixation nearly impossible -10-30 minsufficient; After fixation freely reactive groups need to be blocked: 100mMglycine/PBS or 50mM NH4SO4/PBS or 50mM NH4Cl/PBS.!! Perfect solution for soluble proteins, membrane antigens !!-sometimes cytoskeletal integrity can be compromized.Glutaraldehyde: only a few antibodies like it; blocking of excess with reducingagent NaBH4.EGS (ethylene-glycol-succinimide): preferred for a mix staining of membraneand cytoskeletal structures; rarely used – not well known.