Permeabilization immunofluorescence

Also consistent with this labeling in SERT, antibodies raised against EL2 and EL4 of NET showed reactivity with intact COS cells expressing NET, as assessed by immunofluorescence, whereas antibodies against the amino and carboxyl terminal regions reacted only after cells were permeabilized (21). 

Melan, M. A. and Slnder, G. (1992) Redistribution and differential extraction of solnble proteins in permeabilized cnltnred cells implications for immunofluorescence microscopy. J. Cell Sci. 101, 731-743. 

For cells expressing GFP fusion protein, Hoechst 33342 or Draq5 nuclear stain is added to the fixative to ready the cells for image acquisition. For immunofluorescence staining, the cells are incubated with PBS-TB (PBS with 0.2% Triton X-100, 0.1% bovine serum albumin [BSA]) for 10 min to permeabilize the cell membranes. Primary antibody is then added at 0.5 to 5 pg/mL in PBS-TB and incubated at room temperature for 1 hr or at 4°C overnight. The wells are then washed two times with PBS-TB. Fluorescently labeled secondary antibody is added at 2 pg/mL together with 10 pg/mL Hoechst 33342 nuclear stain in PBS-TB and incubated at room temperature for 1 to 2 hr. The cells are then washed with PBS-TB and once with PBS before image acquisition. 

One major problem area in immunofluorescence is the choice of the primary fixative. For intracellular antigens, unlike surface antigens, the access of a large protein, such as an antibody, requires permeabilizing the cell membrane, and the preservation of cell architecture and antigen distribution after membrane permeabilization requires precise structural... 

Fig. 2. Indirect immunofluorescence labeling of cell-bound component II. Component II (0.5ng/well) was incubated with cultured Vero cells at 37 °C for 7.5 min (a) not permeabilized with Triton X-IOO-PBS and (b) permeabilized with Triton X-100-PBS. For details see text (Section 9.5.4)... 

Type 2 mutant receptors have defective intracellular transport. This phenotype is confirmed by carrying out, in parallel, immunofluorescence experiments on cells that are intact (to demonstrate the absence of cell surface receptors) or permeabilized (to confirm the presence of intracellular receptor pools). In addition, protein expression is confirmed by Western blot analysis of membrane preparations from transfected cells. It is likely that these mutant type 2 receptors accumulate in a pre-Golgi compartment, because they are initially glycosylated but fail to undergo glycosyl-trimming maturation. 

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). 

Cells are grown on cover slips. After fixation they are permeabilized with detergent and incubated in a medium that supports run-on replication. Subsequently, cells are fixed and processed for immunofluorescent labeling. The labeling procedure can be completed in one day. 

The immunofluorescent labeling procedure described in this section is analogous to that described in Section III,A. It involves fixation and permeabilization of the cells and denaturation of the DNA in order to give the antibodies access to the halo atoms of the incorporated IdU and CldU nucleotides. A very similar dual-labeling protocol, more specifically for flow cytometry rather than for fluorescence microscopy, has been described by Aten et al. (1994). 

The development of permeabilized cell systems has facilitated the reconstitution and biochemical characterization of a variety of intracellular protein trafficking steps. In this article I describe how coverslip-attached cells are permeabilized with streptolysin O (SLO) and incubated with exogenously added peroxisomal proteins. The protein import into peroxisomes is analyzed by a simple immunofluorescence technique. The procedure is described for CHO cells but it can be used for a variety of mammalian cells. Furthermore, the system can be applied to study the import of proteins into other subcellular organelles. 

FIGURE I Permeabilization of CHO cells with SLO and reconstitution of peroxisomal protein import. Cells were treated with (A) or without (B) 0.2 U/ml SLO, fixed, and incubated with antitubulin antibodies followed by rhodamine-coupled secondary antibodies. Cells per-meabilized with 0.2 U/ml SLO were incubated for 45 min at 37°C with HSA-SKL (C) or luciferase (D), and the localization of the exogenously added substrates was analyzed by immunofluorescence. Bar = 20 /xm. Reprinted from the Journal of Cell Biology, 1993, Vol. 120, pp. 675-685, by copyright permission of the Rockefeller University Press. 

The quality of immunofluorescence of cellular components (and inununocyto-chemistiy in general) often presents conflicting demands to the investigator the cellular structures should be adequately conserved yet made readily available for antibody labelling, and the reactivity of the cellular epitopes should not be affected. To achieve this, an optimal fixation and permeabilization method should be selected, with the conditions optimized for ... 

Immunolabelling of organelles or stmctiues by fluorescent antibodies in living cells has the advantage, compared to work in ftxed cells, that the dynamics of the labelled structures or molecules can be studied by for example time-lapse microscopy. Also, labelling in living cells does not suffer from possible problems induced by the fixation or permeabilization of the cells as is necessary for immunofluorescence or electron microscopy. 

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