Permeabilization Protocols

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Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 2005 67(1) 4-17. 

The permeabilization protocol with (3-escin is similar to that applied for saponin skinning (Kobayashi et ah, 1989). The pores are quite large, allowing even antibodies to permeate (lizuka et ah, 1994). In these preparations, the pharmacomechanical coupling is functionally intact as judged from the agonist-induced release of Ca + from intracellular stores and increase in Ca + sensitivity of the myofilaments (Kobayashi et ah, 1989). One criterion for complete permeabilization again is the amplitude of maximal Ca +-activated... 

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. 

To investigate the subcellular distribution of internalized receptors, permeabilized cells can be co-stained with organelle-specific antibodies using essentially the same protocol. However, it is essential to ensure that the second reagents do not crossreact. 

This protocol requires fixation and permeabilization of the cells, therefore it is only applicable to ligands for which this is possible. GFP-tagged dominantnegative dynamin constructs are, however, available (19) and should enable the use of live cells. 

Permeabilize the cells with SLO using the protocol described in... 

Two different protocols for the direct labeling of a cell surface antigen are described next one for unfixed cells in suspension and another for fixed cells adhered to coverslips (Fig. 2). A permeabilization step (see Chapter 9) must be added for staining intracellular antigens with fluorescent antibodies or their fragments. 

Drosophila embryos are protected both by an outer layer called chorion and an impermeable and opaque vitelline membrane. Therefore preparation of whole mount Drosophila embryos for staining with antibodies and/or other fluorescent markers must go through the following steps chorion removal, fixation, vitelline membrane removal, and membrane permeabilization. The next subsection introduces the basic procedures for embryo collection and chorion removal that are common to all protocols described here, as well as the two most common fixation methods with or without methanol (the latter requiring hand devitellinization of embryos). The first one works well for immunostaining, while the second is ideal for F-actin staining with phalloidin. 

Key words Flow cytometry, Intracellular protocol, Permeabilization, Fixation, Stain, Antigen... 

A powerful new approach is the direct measurement of released Ca + with fluorescent Ca + indicators such as fura-2 or fluo-3, whereby the permeabilized smooth muscle has to be placed into microcuvettes (lino, 1991). Since force is no longer required as an indicator, Ca + release may be measured in the absence of MgATP, which allowed the study of adenine nucleotides on the IP3-induced Ca + release without the interference by Ca + uptake (lino, 1991 Hirose and lino, 1994). Inclusion of ryanodine allows a separate study of the IP3- and caffeine-sensitive stores (Hirose et al., 1993). Experimental protocols have been designed that allow the use of high concentrations of EGTA, thereby preventing Ca -mediated feedback regulation of the Ca2+ release (Hirose and lino, 1994). Direct measurement of Ca + release with indicators becomes even more powerful in combination with... 

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). 

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