Antibody staining

There are many commercial sources of monodonal antibodies recognizing the halogenated pyrimidines. The choice of antibody often depends on individual preference to one or other company. However, several points should be taken into account as the quality of the measurement will depend on the purity, affinity, and spedfidty of the antibody. For instance high affinity antibodies such as IU-4 (Caltag) will be required if low levels of BrdUrd are to be detected. 

Most antibodies are divalent and their binding is limited when BrdUrd substitution is less than 1%. In some instances it may be preferable to use a rat- 

After denaturation and washing, resuspend the pellet in 0.5 ml of PBS containing 0.5% Tween 20, 0.5% normal goat serum (NGS—this blocking agent can be substituted with 1% BSA), and 20 j,l of rat anti-BrdUrd antibody. Incubate for 1 h at room temperature with occasional mixing. 

Resuspend pellet in 0.5 ml PBS/Tween/NGS containing 20 of goat anti-rat IgG (whole molecule) FTTC conjugate (Sigma Chemical Co.). Incubate for 30 min at room temperature. 

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Van der Bij, W., et al. (1988). Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leukocytes. J. Med. Virol. 25, 179-188. 

Fig. 17.2. (Opposite) Immuno-gold localization of a T. muris-derived IFN-y homologue to the bacillary band and cuticular pore. (A) Transmission electron micrograph of bacillary band showing the pore chamber (PC), pore aperture (PA) and lamellar apparatus (LA) (x22,000). (B) High-power localization of antibody staining (black dots) to the lamellar apparatus (LA) and pore chamber (PC) (x36,000). (Courtesy ofF. Bughdadi.)...

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

S. M. Scholl, C. Pallud, F. Beuvon, K. Hacene, E. R. Stanley, L. Rohrschneider, R. Tang, P. Pouillart, and R. Lidereau, Anti-colony-stimulating factor-1 antibody staining in primaiy breast adenocarcinomas correlates with marked inflammatory cell infdtrates and prognosis, J.Natl. Cancer Inst. 86(2), 120-126 (1994). 

Organotypic, e.g., mimicking skin or mucosa 3-D from 2-D 3-D/in vivo context, imaging using fluorescent cells possible, biological processes can be assessed (invasion, survival, proliferation, differentiation), antibody staining of fixed matrices possible, 24-weU format Slow (8-day contraction of 3-D matrix), invasion 0-21 days, proliferation not excluded, laborious, cannot be used in HT (31)... 

Fig. 4.5.4 Identification of mutations in the transferrin protein by neuraminidase treatment. Unusual patterns in the IEF of serum transferrin might lead to pitfalls in CDG diagnostics. These varying patterns are often due to mutations of charged amino acids in the protein backbone of the transferrin molecule, which might lead, for example, to an accumulation of trisialo transferrin bands (lane 3, indicated by a question mark). Further investigations are carried out by cleaving off charged sialic acid monosaccharide moieties from transferrin-linked oligosaccharides by neuraminidase treatment, followed by IEF and transferrin antibody staining. In the case of protein mutations, additional bands below (lane 4) or above (not shown) the desialylated transferrin form appear...

Fig. 4.5.10 Analysis of core-1 mucin type -linked glycans derived from apolipoprotein C-III (ApoC-III). Serum-derived ApoC-III from a control (lane 1, left) and a CDG-IIx patient (lane 2, right) was investigated by IEF followed by antibody staining with a polyclonal rabbit-a-human ApoC-III antibody. ApoC-IIfi ApoC-IIEand ApoC-III0 indicate the variability in the amount of sialic acid residues linked to ApoC-III...

A similar method is used for beads with defined fluorochrome content (e g, Quantum beads, Flow Cytometry Standards Corporation), except that the beads are not subjected to the antibody-staining procedure The number of fluorochrome molecules per cell is deduced from the standard curve, and is converted to bound antibody molecules from knowledge of the fluorochrome protein ratio of the conjugate. 

Antibody staining buffer PBS containing 0 5% (v/v) Tween-20 and either 1% (w/v) BSA (for direct antibody conjugates), or 0 5% (v/v) normal goat serum when a second antibody is used. 

Resuspend pellet in 90 pL of antibody staining buffer containing BSA (Section 2), and add 10 pL (see Note 7) of MAb The recommended antibodies are PC 10-FITC (DAKO F863) and Ki-67-FITC (DAKO F788). The appropriate directly conjugated isotypic control antisera are also available from DAKO The suspensions are incubated for 1—2 h at room temperature and protected from light... 

Linden, M. D., Nathanson, D., and Zarbo, R. J. 1994. Evaluation of anti-p53 antibody staining Quality control and technical considerations. Appl. Immunohistochem. 2 218-224. 

Other types of calibration help may be available in the form of a different type of calibrated microsphere. Beads can be obtained that possess a known number of binding sites for immunoglobulin molecules. They can be treated as if they were cells and stained in the routine way with the antibody stain (direct or indirect) in question. The intensity of the beads with known numbers of antibody binding sites can be used to calibrate the scale, converting ADC channels to... 

Fig. 1. A high-throughput platform of the carbohydrate-based microarrays. A high-precision robot designed to produce cDNA microarrays was utilized to spot carbohydrate antigens onto a chemically modified glass slide. The microspotting capacity of this system is approximately 20,000 spots per chip. The antibody-stained slides were then scanned for fluorescent signals with a Biochip Scanner that was developed for cDNA microarrays. The microarray results were subsequently confirmed by at least one of the conventional alternative assays.

For enzyme inhibition assays, urine is the preferred specimen [4]. Interestingly, Bik can be measured by the inhibition of trypsin in urine but not in plasma. Urinary Bik analysis may also be performed by antibody staining, latex agglutination, and radioimmunoassay (RIA) [4]. Despite the analytical approach used, all Bik forms are measured together. The enzyme inhibition method involves adding known amounts of trypsin to the specimen and monitoring trypsin inhibition. Trypsin activity is assessed by detection of by-products from a cleavable substrate. Dipstick methods are available for the rapid detection of trypsin inhibitors in urine [15, 17 19]. 

Figure 3 Quantifying nuclear trafficking - Experimental ratios of the amount of NF-kB antibody staining in the nucleus as compared to the cytoplasm, for unstimulated and stimulated (10 ng/ml TNF-a) HeLa cells. The Z-factor for the assay was 0.73. The CV was 6.3%...

FITC-conjugated anti-BrdU antibody staining solution Make a solution of PBS + 0.1% Triton X-100 + 1% BSA + 0.1 pg/mL FITC-conjugated anti-BrdU antibody. This is made up freshly for each experiment and kept on ice. 

FIG. 5. (Left) Pan Na channel antibody staining of control and HSV-infected (24 h) DRG neurons (top, whole cell staining bottom, cross section image). (Right) Quantified immunofluorescence of Na channel antibody staining. 

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