Immunostaining antibodies

Key words Microtubules, Immunostaining, Antibody, Fluorescent dye. Super-resolution microscopy. Stochastic activation... 

Fig. 4.2 Dorsal root ganglia pathology in HIV neuropathy is characterized by foci of macrophage-lymphocytic infiltration (a) and Nagoette nodules (b). Infiltration by activated macrophages is demonstrated by immunostaining with anti-CD68 antibodies (c) (scale bar, 50 pm). Reproduced with permission of Wiley-Blackwell Pubhshing (Pardo et al. 2001)...

Fetsch PA, Abati A. The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer a query and a review of the literature. Diagn. Cytopathol. 2007 35 319-328. 

Standardization of IHC/ICC has been a critical issue for more than three decades, especially with the advances in targeted therapy such as the development of trastuzumab (Herceptin) for advanced breast cancer.51 Nevertheless, standardization is a difficult issue because numerous factors may influence the consistency and reliability of immunostaining results, including fixatives, fixation time, AR, antibody clones, detection system, and interpretation (see Part II). In cytopathology, the situation is even worse due to its variable cell sample preparation techniques. Cytopreparation is. .. the foundation of cytomorphology. 52 We believe it is also the foundation of ICC. Therefore, standardization of ICC needs to start with uniform and reliable cytopreparation. 

Figure 16.1 Montage of images, after immunostaining of peptides. The antibody clones for these analytes are D07 (p53), 9C2 (HER2), 1D5 (ER), and 636 (PR). The peptides were spotted in duplicate, adjacent to each other. The left-hand column ( Not Fixed ) illustrates stained peptide spots that were not fixed, representing a baseline condition. The middle column was fixed in formalin and not antigen retrieved. The peptides for p53 and HER2 lost immunoreactivity whereas the peptides for ER and PR continued to be immunoreactive. The right-hand column of peptide spots were both formalin fixed and antigen retrieved. Adapted with permission from Reference 16, 2004 American Society for Clinical Pathology.

Figure 16.5 Immunostained peptide arrays after various treatments of fixation, protein cross-linking, and antigen retrieval, as indicated at the top. Each row has a different peptide that is immunoreactive for the antibody denoted to the left. Column A represents the baseline condition, without any treatment whatsoever. Column B shows immunoreactivity of each peptide after overnight formalin fixation. Column C shows the immunoreactivity after first coating the array with an irrelevant protein (casein) followed by overnight formalin fixation. Column D illustrates the immunoreactivity of the peptides after the treatment of column C, and then antigen retrieval. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

Further dilute (if required) the resultant primary antibody Fab fragment complexes to optimal working concentration (usually about 1 5 pg/ml) in staining buffer containing 10% normal serum and then apply to the sample for 30 60 min at room temperature and proceed further with your standard immunostaining protocol. 

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