Immunostaining fixatives

Suthipintawong C, Leong AS-Y, Vinyuvat S. Immunostaining of cell preparations a comparative evaluation of common fixatives and protocols. Diagn. Cytopathol. 1996 15 167-174. 

Figure 5.2 Comparison of immunohistochemical staining results among variable periods, 6 h to 30 days, of formalin-fixed, paraffin-embedded human breast cancer tissue (A-N), and cell line MCF-7 sections (O-Bl). All four markers, estrogen receptor (ER) (A-G), CK (cytokeratin cocktail, H-N), Her2/neu (O-U), and MIB-1 (V-Bl), showed comparable positive immunostaining results at +++ level after antigen retrieval. Original magnification x 200. Bar = 50 tm. Reproduced with permission from Shi et al., I Histochem. Cytochem. 2007 55 105-109. See color insert.

Figure 7.10 Immunostain result with inadequate antigen retrieval, resulting in staining of unfixed but not fixed peptide controls. The HER2+ tumor (left slide) is largely unstained as well. See color insert.

Even though AR was originally designed for FFPE, several investigators found it can improve immunostaining on smears fixed in alcohol, formalin, Carnoy s Pap, and ThinPrep fixative.27,48 Gong et al. compared a number of... 

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.

Notes. Mouse tissues were fixed with 4% formaldehyde for 6h. Paraffin sections were boiled in 20 mM Tris-HCl buffers (TB), at pH 6.0 or at pH 9.0, for 10 min. After cooling, the sections were briefly washed with distilled water and heated in another buffer (e.g., pH 9.0 and then 6.0) for 5min. Some specimens treated in the second buffer were heated in the first buffer (e.g., pH 9.0, then pH 6.0, and finally pH 9.0 pH 9-6-9) for 5 min. The antigens were localized in respective tissues described in Table 17.1. Immunostaining was scored as follows +++, strong ++, moderate +, weak , faint -, negative. 

Imam SA, Young L, Chaiwun B, et al. Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissue for immunostaining. Anticancer Res. 1995 15 1153-1158. 

Xiang, C.C. et al. (2004) Using DSP, a reversible crosslinker, to fix tissue sections for immunostaining, microdissection and expression profiling. Nucleic Acids Res. 32, el 85. 

Figure 6. Cytoskeletal proteins in nucleus. HeLa cells were immunostained with anti-keratin 8 or anti-keratin 18 antibodies, (a) Paraformaldehyde-fixed cells, (b) Cells treated with detergent before the fixation. (Scale bars 10pm)...

Figure 7. A series of monoclonal antibodies raised against nuclear proteins. HeLa cells fixed with 4% paraformaldehyde were immunostained with monoclonal antibodies raised against nuclear proteins. The intracellular localization of these antigens are (a) dot inside the nucleolus, (b) whole nucleolus, (c) nuclear foci, (d) nucleoplasm, (e) the edge of the nucleus, (f) cytoplasm, (g) cytoskeleton, (h) plasma membrane, (i) mitochondria, (j) nucleus and cytoplasm, (k) nucleus and the paranuclear structure, and (1) paranuclear structure and nucleoplasm...

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