Fixation and staining

Immunohisto- chemistry 10-100 mg 1-2 pm Boron must stay during fixation and staining Antibodies must be available 20 ppm yes no ... 

After the run common fixation and staining protocols are used. For Western blotting according to the Kyhse-Anderson semidry protocol (Protocol 2.4.3), the following buffers are recommended by ScHAGGER and v. Jagow ... 

The authors describe the same detection hmit if compared with Coomassie, hut in our opinion, copper staining is less sensitive and has the main disadvantage that it is impossible to dry copper-stained gels. On the other hand, proteins are not denatu-rated by fixation and staining and can be eluted from gels in high yield. 

Published immunohistochemical studies of overexpression of HER-2 report a wide range of overexpression rates, ranging from 9 to 60%. The likely reason for this variation is the use of different antibodies, tissue types, and fixation and staining protocols. In addition, different scoring systems result in interpretation differences. To minimize these variations the use of a uniform immunohistochemical method, such as the HercepTest, is recommended. The HercepTest kit provides standardized procedure and evaluation criteria. 

Protocols similar to Method 2 are used Antibodies are first bound to unfixed cells at 4°C, and the cells warmed to 37°C in the presence or absence of chemokine. At the required times, the cells are cooled to 4°C, fixed, and stained with specific fluorescent second reagents either intact or following permeabilization. Alternatively, the cell-surface antibodies can be removed by acid elution before fixation and staining. 

We use an ELX405 plate washer (BioTek) running at lowest dispensing and aspiration speed to assist all fixation and staining processes. A concentrated fixative—either formaldehyde in phosphate buffered saline (PBS) or Mirsky s fixative (National Diagnostics)—is added directly to the wells. After 15 to 60 min, the wells are washed once with PBS. 

Selective monitoring using a suitable wavelength makes the detection of amphoteric compounds other than high-molecular-weight proteins possible, since no fixation and staining procedures are necessary. Since most ampholytes absorb strongly below 280 nm, low-UV detection is usually not possible in CIEF. 

Fig. 3.5. Scatter assay. In the illustrated example, B16 melanoma cell colonies have been allowed to grow from few cells either in the absence (control) or in the presence (40 ng/ml) of HGF/SF. After fixation and staining with crystal violet, representative colonies have been photographed.

Strobel, S., Miller, H.R.P. and Ferguson, A. (1981). Human intestinal mucosal mast cells evaluation of fixation and staining techniques. J. Clin. Pathol. 34, 851-858. 

Zalokar M, Erk I (1977) Phase-partition fixation and staining of Drosophila eggs. Stain Technol 52 89-95... 

Which technique is used depends, to some extent, on what is required from the observation. For example, during processing by conventional methods, virtually all elements such as sodium and potassium are removed. Therefore, X-ray analysis for these elements would be useless however, the morphology is totally acceptable. Other elements may survive the processing and the only consideration for X-ray analysis is interference of overlapping peaks due to the fixation and stain. 

ESCA measurements ESCA(Electron Spectroscopy for Chemical Analysis) spectra were obtained on a Shimazu ESCA Model 650-B electron spectrometer using MgKa radiation. Samples of membranes cast from chloroform (Cf) - trifluoro-ethanol (TFE) 10 1 mixture, followed by fixation and staining of membranes by osmium tetroxide (OsO ) were coated onto plane glass plate, and fixed to the probe by doublesided tape. 

Thin sections are prepared by ultramicrotomy after special fixation and staining procedures. Investigations are carried out by conventional TEM, high-voltage TEM, or AEM. 

Moreover, the morphology of specimens can be stabilized by applying chemical fixation and staining treatments, essentially by cross-linking the macromolecules and incorporating atoms of heavy elements. On the other hand, it is possible to make use of the irradiation sensitivities of polymers for special effects for contrast development see Section 2.2.4. 

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