Compromises between penetration of antisera and preservation of ultra structure in pre-embedding electron microscopic immunocytochemistry.
Light microscopic pre-embedding immunocytochemistry on free-floating brain sections is commonly performed with antisera to which a detergent is added. For instance, the trajectories and the terminals of axons of neurons labelled by anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L) are commonly visualized by immunocytochemistry, using antisera to which 0.5 per cent Triton X-100 is added. In this material the ultrastructural details are very poorly preserved. Omitting the detergent from the antisera results in no or very superficial staining. To improve the quality of ultrastructural preservation while still obtaining sufficient penetration of the antisera, we subjected vibratome sections of rat brain containing PHA-L-labelled neuronal perikarya and processes to PHA-L-immunocytochemistry in the presence, at various concentrations, of the detergents Kodak Photo-Flo 200, Triton X-100, Saponin, Nonidet P-40 or sodium deoxycholate. As controls served incubations without detergents. The addition of 0.1 per cent Photo-Flo 200 to all the immunoreagents, together with a relatively long incubation time, results in a balanced compromise between the demands of preservation of the ultrastructure and penetration of the antisera. If the PHA-L-immunocytochemistry is preceded by a short treatment with 0.05-0.1 per cent Triton X-100, the penetration is further improved, although at the expense of ultrastructural detail.
Wouterlood
F G
FG
Department of Anatomy, Vrije Universiteit, Amsterdam, The Netherlands.
Sauren
Y M
YM
Pattiselanno
A
A
D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D003902:Detergents; D005260:Female; D007106:Immune Sera; D007150:Immunohistochemistry / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D017830:Octoxynol; D010835:Phytohemagglutinins / Q000276:immunology*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D013501:Surface-Active Agents
D003902:Detergents; D007106:Immune Sera; D010835:Phytohemagglutinins; D013501:Surface-Active Agents; C100156:leukoagglutinins, plants; D011092:Polyethylene Glycols; D017830:Octoxynol