Extraction of label during processing of brain tissue for electron microscopic autoradiographic investigation of glycoprotein metabolism.
Rats were injected intracerebrally with (3H) fucose and survived for 30 min and 24 h. Following perfusion fixation with aldehydes, Vibratome sections of the brains were processed for embedding in resin and the radioactivity in the various solutions measured by scintillation counting. The decline of radioactivity was monitored in up to 18 successive buffer washes. Whereas 85% of the initial radioactivity could be eluted after 30 min, only 7% were extractable after 24 h survival time. Addition of unlabelled L-fucose to the fixative and buffers caused a small insignificant increase of the total radioactivity extracted. Only small amounts of radioactivity could be removed by treatment with trichloroacetic acid after extensive rinsing in buffer. It is concluded that thorough rinsing in buffer (which is more important with short-time experiments) is effective in removing the acid-soluble radioactivity. An additional safe-guard lies in dehydration by ethanol. The ethanol series also removes acid-soluble radioactivity in addition to small amounts of what is probably higher molecular weight material.
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002021:Buffers; D005643:Fucose; D006023:Glycoproteins / Q000378:metabolism*; D006652:Histological Techniques; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D012588:Scintillation Counting / Q000379:methods; D013997:Time Factors; D014316:Tritium
D002021:Buffers; D006023:Glycoproteins; D014316:Tritium; D005643:Fucose