Fixation of cells

Fixation of cells in l% formaldehyde in PBS will change their light scatter characteristics slightly. Therefore, when comparing samples over a time-course, it is important that all samples should be fixed for at least 24 h before analysis. 

The latter is dependent on interplay between the degree of permeability of the cell/tissue and the size of the probe. In practice, probes with a median size of 200-400 bp are suitable for cells/tissues fixed in aldehyde and subjected to varying degrees of proteolysis (see Section 3.). I have found that fixation of cells m aldehyde followed by mild proteolysis produces the best results. 

Common procedures to obtain permeabilization and fixation of cells in enzyme immunohistochemistry by light microscopy (adapted from Jessen, 1983) ... 

Most immunofluorescence protocols involve fixation of cells at a specific time point of activation to preserve the distribution of intracellular proteins, followed by cell permeabilization to allow the entry of the antibody into the cell. The primary antibody can either be directly tagged with a fluorophor or, in the indirect approach, a secondary anti-lgC or anti-lgM antibody is tagged. The direct approach is simpler and less prone to background problems. In contrast, the indirect approach offers the advantage of widely available labeled antibodies and the increased sensitivity that results from amplification of the signal (for more details, see Harlow and Lane, 1988). 

Fixation of Cells Grown in Culture on Microscope Cover Slips... 

Figure 1 shows time courses of changes in photosynthetic C0 -fix-ation and the carboxylase activity of RuBisCO of Euglena cells(6 during the cell cycle in synchronized cultures. The carboxylase activity of RuBisCO extracted from those synchronized cells increased nearly double during the growth phase and decreased during the division phase(Fig. 1, middle). The photosynthetic C02 fixation of cells, measured at the same... 

In this work, an adequate concentration of glutaraldehyde used for fixation of cells before DAPI-staining was selected, because the concentration was found to affect profoundly the shape and distribution of cp-nucleoids. 

The initial fixation of cells to the slide by heat should be done with two to - three quick passes of the slide over the flame of a bunsen burner and not... 

Fixation of cells by paraformaldehyde is used when cell surface staining is re quired. It is also a good choice for labelling of microfilaments, although microtubules are not fixed well by it. 

Brightfield microscopy. Unstained cells have little contrast with their surroundings and may be difficult to see. Therefore, samples are typically stained to increase contrast between the specimen and the background. Samples can be treated with dyes that bind selectively to the whole cell or to certain cellular components to increase contrast. However, most staining techniques kill cells and fixation of cells to the slide is usually required. These techniques may also alter the structure of the specimen. 

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