API Publ 4647:1997 pdf download

API Publ 4647:1997 pdf download.BRAIN GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP) AS AMARKER OF NEUROTOXICITY DURING INHALATION EXPOSURE TO TOLUENE.
Although most of these points will be familiar to experienced researchers, these points would be especially valuable to laboratories setting up to run this assay.
Tissue preparation. The dissection criteria are important. A training session and inter-lab comparisons should be conducted periodically. Unusual tissue weights may reveal dissection error.
Total protein. Glassware cleaning must receive normal degree of care. Glassware should be soaked in soap overnight then carefully washed to eliminate all traces of protein. The microtiter plates must be handled carefully to avoid contamination with extraneous protein. Incomplete sonification of brain tissue can be a problem, causing minute specks of intact tissue to be suspended, potentially overloading a well. Total protein results should be scrutinized to confirm they are within the range of values which are typical for each brain region, both from the labs own historical data and from the published literature, then subject to statistical analyses to determine if there are significant differences between batches of control specimens, and if there are significant effects related to toxicant dose.
GFAP. It is preferable to prepare multiple aliquots of each brain homogenate immediately at the time the animal is sacrificed, so as to allow for the assay to be repeated as a repLication and quality control without repeated thawing, as would be the case if only one aliquot were prepared. However, the multiple aliquots are costly, both in staff time and in the greater amount of freezer space required. The OFAP assay should be set up so a number of samples can be completely assayed in one long working day; an experienced technician can complete 8 plates (each with 40 samples in duplicate). In order to minimize staff scheduling problems. sample dilutions may be prepared and total protein determined in brain specimens the day before the GFAP assay; sample dilutions for GFAP assay may be kept overnight at -80C, then thawed for the OFAP assay the next morning. Other schemes for breaking up the assay into 2 days’ work have been less successful, inducing variability. Selection of the standard data is important; an internal standard.