Monoclonal antibodies staining cells with

Tomehave, D., Hougaard, D. M., and Larsoon, L.-L. 2000. Microwaving for double indirect immunofluorescence with primary antibodies from the same species and for staining of mouse tissues with mouse monoclonal antibodies. Histochem. Cell Biol. 113 19-23. 

The correct control is always an antibody of exactly the same properties as the monoclonal antibody used in the experiment, but with an irrelevant specificity. If we are staining cells with a monoclonal antibody having a specificity for the CD3 protein occurring on the surface of T lymphocytes (and that monoclonal antibody happens to be a mouse immunoglobulin of the IgG2a subclass, conjugated with six fluorescein molecules per molecule of protein and used to stain the cells at a concentration of 10 pg per ml), then an appropri-... 

Immunohistochemical staining of Bacillus anthracis with monoclonal antibodies against cell wall and capsule antigens has been successfully used in the recognition of bioterrorism-related anthrax cases and is an important step in early diagnosis and treatment (Fig. 3.26A-C). Gram staining and culture isolation of... 

The major mature blood and immune cell types (erythrocytes, granulocytes, monocytes, and lymphocytes) are easily identifiable by microscopic observation of histochemically stained cell preparations, but distinguishing between mature cell subclasses such as B and T lymphocytes requires the use of monoclonal antibody staining for cell surface proteins in combination with multicolor flow cytometry (discussed later). 

Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. 

Nielsen, M.H., Bastholm, L., Chatterjee, S., Koga, J., and Norrild, B. (1989) Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections. Elistochemistry 92, 89-93. 

Light and electron microscopic studies were performed on the synovial membranes (E2) of patients with HIV associated arthropathy. An immunoperoxidase technique with the use of monoclonal antibodies against CD4, CD8, B, and DR lymphocytes and HIV p 24 antigen was also used. Mild to moderate nonspecific proliferative changes and increased vascularity of the subsynovial space were seen. Immunohistochemical staining revealed HIV p 24 positive staining cells of the synovial lining layer and the mononuclear cells of the subsynovial space, CD4, CD8 with predominance of CD8, B, and DR cells were also present. 

Figure 7 Immunopotentiating reconstituted influenza virosomes (IRIV) mediated adjuvance in cytotoxic T-cell induction requires CD4+ T cells. CD8+ and CD14+ cells were cultured in the presence of autologous intact or irradiated CD4+ cells. These cultures were stimulated with influenza matrix (IM)58 66 (1 Pg/mL) alone (A) or supplemented with IRIV (1 50) (B). After seven days of incubation both cocultures were restimulated with irradiated IMss-ee pulsed CD14+ cells and cultured for six further days in the presence of interleukin-2 [see Materials and Methods ]. Six days after restimulation, cultures were stained with HLA-A0201 /IM58-66 PE-specilic tetramers and anti-CD8 fluorescein isothiocyanate monoclonal antibodies. Source. From Ref 6.

It is entirely possible that surface staining cannot be accomplished before fixation. Some antibody-antigen complexes cannot withstand chemical fixation and/or permeabilization. An empirical evaluation must be made. In this example, cells are first stained with a monoclonal antibody against a cell-surface receptor, fixed with ethanol, and then the DNA is stained with propidium iodide. The cells are analyzed for two-color fluorescence, the green of the fluorescein-labeled surface marker and the red of the labeled DNA intercalator. This approach works for antibody-antigens that are unaffected by fixation. 

Stain the cells by adding the appropriate amount of the primary monoclonal antibody diluted in PBSG with 10% bovine serum albumin (see Note 2). 

Qualitative analysis of intrahepatic distribution is possible with immunohistochemistry. With antibodies against the carrier or the carrier-bound drug this compound can be localized in liver sections [152],To identify the cell type(s) involved in the uptake, the sections can subsequently be double stained with markers for the different cell types. In the rat liver, the monoclonal antibodies HIS52 (anti-rat endothelial cell antigen-1 or anti-RECA-1), ED2, the combination of anti-desmin and anti-glial fibrillary acidic protein, and anti-aSMA are generally used to identify SECs, KCs, quiescent HSCs, and activated HSCs, respectively. 

The actual response of monoclonal antibodies with individual cells is usually visualized either directly (typically using fluorescent stains) or indirectly [using the reaction of antibody labeled with horseradish peroxidase (HRP) or other enzymes] with diaminobenzidine (DAB) (or other substrate while using other enzymes) under the microscope or in the flow cytometer. The latter, however, is not employed routinely in CSF immunocytology, although it has an advantage in clinical hematology. 

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

The immunocytochemical staining of cytochrome C offers another alternative since, upon exposure to apoptotic stimuli, cytochrome C is rapidly released into the cytosol, an event that may be required for the completion of apoptosis in some systems (L2). The effect of cytosolic cytochrome C is thought to be the activation of caspases. The immunocytochemical staining of cytochrome C localized in mitochondria in healthy cells or diffused in the cell cytoplasm with monoclonal antibody (Promega) after induction of apoptosis, as detected by fluorescence microscopy, can be used for monitoring apoptosis (L2, M7, S8, T6). It is also a simple, rapid, specific mefhod for quanfifafive assessment of apoptotic cells. 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

Analyze on a flow cytometer (see Notes 9 and 10). This type of staining can be analyzed on any of the modem flow cytometers with the proviso that the machine is equipped with a pulse processing facility to enable the discrimination of cell doublets. The most commonly used flow cytometer is the Becton Dickinson FACScan. In this machine, PI should be collected into FL3 rather than FL2 to overcome any crossover of the FITC into the FL2 channel The FL3 detector should be routinely set around 400, whereas FL1 is usually set at around 500 in linear amplification Controls, without either BrdU or the monoclonal antibody, should be included whenever possible to determine the lower limits of detection of the DNA precursor. At least 10,000 events should be analyzed, but more might be required in the case of slowly proliferating (low BrdU incorporation) tissues or tumors. 

---