ABSTRACT
Objective:To establish a method for the determination of oleanolic acid in She medicine Clematis florida var. plena. Methods:The HPLC method was carried on a Lanbo-Kromasil C18 chromatography column using acetonitrile-water ( 86∶ 14 ) as the mobile phase. The detection wavelength was 205 nm, the flow rate was 1. 0 ml·min-1 , and the column temperature was 25 ℃. Re-sults:The calibration curve of oleanolic acid was linear within the range of 0. 52-15. 60μg (r=0. 999 9). The average recovery of oleanolic acid was 92. 13%(RSD=3. 0%, n=9). Conclusion:The method is simple, rapid, accurate and reproducible, which can be used as an ideal method for the quality control of She medicine Clematis florida var. plena. .
ABSTRACT
The cytoskeleton of BT_(325), a human glioma cell line, was studied by immunofluorescence microscopy. The effects of different fixatives and buffers on microtubules, microfilaments and intermediate filaments were also compared. It was observed that besides microtubules and microfilaments all cells expressed vimentin. However, only a small fraction of the cells were GFAP positive. We conclude that vimentin is the main component of the intermediate filaments in BT_(325) cells. It was also observed that methanol fixation and formaldehyde fixation followed by acetone or Triton X-100 treatment gave rise to satisfactory results for microtubule immune-staining, and methanol or acidic alcohol fixation resulted in bright staining of intermediate filaments. Formaldehyde fixation also resulted in excellent staining for mierotubnles, but weaker staining for intermediate filaments.If the cells were immune-stained after treatment with Triton X-100, the composition of the buffers had profound effects on microtubules and intermediate filaments, but less effects on microfilaments.It was also observed that if Triton-treated ceils were fixed with formaldehyde before immunostaining with monoclonal antibodies to vimentin, the staining was very weak. However, if the ceils were immunostained first and then fixed with for-maldehyde the staing was quite bright. We conclude that for-maldehyd e fixation may "mask" or destroy certain epitopes on vimentin molecules.
ABSTRACT
Cells of BT_(325), a human glioblastoma cell line, were treated with 0.5% Triton X-100 and immunostained with a monoclonal antibody to vimentin and antiserum to glial fibrillary acidic protein (GFAP), and followed by fluorescence labeled secondary antibody. After observation with fluorescence microscope the cells were dehydrated in increasing grades of ethanol and then critical point-dried. The carbon-platinum coating replicas of cytoskeleton were observed by transmission electron microscopy. The morphology of whole cytoskeleton as well as the arrangement of microtubules, microfilaments and intermediate filaments were clearly observed by this technique. It was observed that the diameter of intermediate filaments highly increased in the cells stained with the monoclonal antibody to vimentin. In the samples stained with anti-GFAP intermediate filaments were decorated in some cells but undecorated in other cells. The results indicates that all BT_(325) cells express vimentin, but only a fraction of the cells express GFAP. It was also observed that if the cells were fixed with formaldehyde before immunostaing with the monoclonal antibody to vimentin, the diameter of the intermediate filaments only slightly increased. However, the diameter of the intermediate filaments highly increaed in the cells stained firstly with the monoclonal antibody to vimentin and followed by formaldehyde fixation. This result is in accord with that of our previous immunofluorescence study. It indicates that formaldehyde fixation may "mask" certain epitopes on vimentin molecules.