Antibody penetration

As explained in this chapter, glutaraldehyde introduces mostly irreversible protein crosslinks that may alter the conformation of the antigen (epitope) molecule. Such extensive crosslinkages become a barrier to the antibody penetration, and thus its accessibility to the epitope is hindered. This impediment becomes a serious problem when monoclonal antibodies specific for only one epitope type are used and/or when epitopes are not located at the surface of the antigen molecule. 

It should also be noted that denaturing agents make cells permeable, facilitating antibody penetration. Moreover, these agents are used usually in combination with microwave heating. Therefore, the role of these agents in epitope retrieval needs to be explained in the context of their role in cell permeabilization as well as of the influence of elevated temperatures. 

Treat the sections with a mixture of 3% normal serum and 0.4% Triton X-100 for -30-60 min at room temperature to aid antibody penetration and block background staining. 

Mechanism of Anti-dsDNA Antibody Penetration into Cells... 

A6. Alarcon-Segovia, D., and Llorente, L., Antibody penetration into living cells. IV. Different effects of anti-native DNA and anti-ribonucleoprotein IgG on the cell cycle of activated T gamma cells. Clin. Exp. Immunol. 52, 365-371 (1983). 

V8. Vlahakos, D., Foster, M. H., Ucci, A. A., Barrett, K. J., Datta, S. K., and Madaio, M. P., Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo. J. Am. Soc. Nephrol. 2,1345-1354 (1992). 

Fig. 4. Immunocytochemistry of formalin fixed 147L) cell cultures using a-PR-A anti-receptor MAb (left) and a control MAb (right). Immunocytochemistry was performed with T47D breast cancer cells grown as monolayers in chamber slides. Cells were fixed for 15 min with 3.7% buffered formalin, followed by a permeabilization step with Triton X-100 (0.1%) for antibody penetration into the cell. Immunocytochemistry was performed by the indirect avidin-biotin immunoperoxidase method using diaminobenzi-dine as the chromagen.

Numerous studies have tried to elucidate the role of onconeural antibodies in other PNS. Immunoglobulin G (IgG) containing Hu antibodies penetrated viable neurons expressing the Hu antigen in in vitro experiments [185], When the IgG fraction of Yo antibody positive sera is administered intraperitoneally in laboratory animals and the blood-brain barrier is disrupted, intracellular IgG can be demonstrated in Purkinje cells [186]. Intrathecal antibody production takes place in many of the PNS [159, 187], and autopsy studies of affected individuals show deposits of IgG in neurons [80, 188, 189] and in the tumor [189]. These results all favor a pathogenic role for the onconeural antibodies. 

Key words Cytocentrifuge, Cells, Smears, Touch preparations/imprints, DMSO, Fixation, Antibody penetration, Charged slides... 

Key words Cell culture, Fixation, DMSO, Antibody penetration, Cell pellets... 

Permeabilize Tissue and Cells to AUow Antibody Penetration... 

Effects of Blocking Agents on Antibody Penetration. Combined Incubation Step. 

The r antibody incubation can be as short as 6 h or as long as 24 h at a dilution determined by preliminary experiments with fluorescent 2° antibody. All incubations should be done on a rotator to maximize antibody penetration. At the end of the 1° antibody incubation, perform 4-6 rinses in buffer with blocking reagents for 10 min each. 

Embedding the tissue with standard hydrophobic epoxy resins will not allow antibody penetration, even to the edge of the tissue section. Two approaches are used to circumvent this problem. First, tissue sections with a standard resin are treated with 10% H2O2 to etch the epoxy section on the sections cut surface allowing antibodies to contact proteins in the section. Fixation can be with paraformaldehyde or... 

Floating section immunocytochemistry - performed on free-floating, thick section (25-100 mm) cut on a Vibratome or freezing microtome with antibodies penetrating deeper because of the greater movement of the section floating both sides of the section will be exposed to the antibodies. 

Consideration of the structure and distribution of cannabinoid receptors is critical to the successful application of immunocytochemical approaches to the identification of cannabinoid receptors in cells and tissues. CBi and CB2 receptors exhibit greatest type specificity at their extracellular amine-terminal domains. Thus, experiments designed for identification of cannabinoid type-specific receptor expression at the protein level should entail use of affinity-purified antibodies directed against the amine-terminal domains. Use of such antibodies is also critical for the identification of cell surface expression of cannabinoid receptors. On the other hand, if assessment of expression of fully processed receptor protein is required, then antibodies directed against carboxy-terminal domains of the CBi or CB2 should be considered. Immunocytochemical methods in which such antibody preparations are considered should include a limited cell/tissue solubilization so as to allow for adequate antibody penetration. A list of commercially available antibodies to domains of CBi and CB2 receptors is included in Table 1. In addition, various laboratories have applied these commercially available antibodies (or have developed their own antibodies) to the identification of cannabinoid receptors at the fluorescence, light, and electron microscopy levels. Table 2 lists references in which immunocytochemical approaches have been utilized for the identification of cannabinoid receptors in a variety of tissues and cell types. 

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