Journal of Histochemistry and Cytochemistry Priciples for Free Access to Science
  Search:   
    >> Advanced Search

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact
This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by VANHA-PERTTULA, T.
Right arrow Articles by GRIMLEY, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by VANHA-PERTTULA, T.
Right arrow Articles by GRIMLEY, P. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

LOSS OF PROTEINS AND OTHER MACROMOLECULES DURING PREPARATION OF CELL CULTURES FOR HIGH RESOLUTION AUTORADIOGRAPHY QUANTITATION BY A MICROMETHOD

TAPANI VANHA-PERTTULA 1 and PHILIP M. GRIMLEY 1

1 Endocrinology Branch and Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014

A reproducible procedure was devised in order to measure the extraction of 3H-labeled cellular products during aldehyde fixation and subsequent processing for electron microscopic autoradiography. Human carcinoma cell monolayers were cultivated in combustible plastic wells, so that all label could be counted as 3H2O. Radioisotope extraction during individual steps of processing could be analyzed and separate groups of experiments were directly comparable. Initial aldehyde fixation and subsequent buffer washes caused the major loss of radiolabeled amino acids, but this never exceeded 15% under conventional conditions. Radioisotope losses were influenced by the relative duration of fixation and buffer washes, fixation temperature and fixative concentration. Formaldehyde and glutaraldehyde both produced a nonspecific, time-related binding effect when 3H-labeled amino acids were introduced along with the fixative. Less significant nonspecific binding was observed when 3H-mannose, 3H-uridine or 3H-thymidine was added. Extraction of radioisotopes during formaldehyde fixation of cell cultures labeled with protein precursors was consistently greater than during glutaraldehyde fixation. Differences were less marked with the other precursors. Evaluation of the total protein extraction is complex, since the net losses observed were apparently the sum of precursor extraction, nonspecific amino acid binding and real molecular extraction. The implications for quantitative interpretative interpretation of high resolution autoradiography must be considered.

Submitted on February 9, 1970


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
J. Serth, M. A. Kuczyk, U. Paeslack, R. Lichtinghagen, and U. Jonas
Quantitation of DNA Extracted after Micropreparation of Cells from Frozen and Formalin-Fixed Tissue Sections
Am. J. Pathol., April 1, 2000; 156(4): 1189 - 1196.
[Abstract] [Full Text] [PDF]



Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact
The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 1970