Staining, immunohistochemical

Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 1991 39 741-748. 

Maleszewski J, Lu J, Fox-Talbot K, et al. Robust immunohistochemical staining of several classes of proteins in tissues subjected to autolysis. J. Histochem. Cytochem. 2007 55 597-606. 

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.

The advent of personalized therapies that are dependent on the outcome of an immunohistochemical stain has increased the need for quantitative positive controls. When IHC was first introduced as an adjunct in surgical pathology diagnosis, the interpretation was largely qualitative. Specific markers were present or absent, thereby characterizing a tumor cell s lineage. The fact that IHC interpretation was qualitative, rather than semiquantitative, minimized... 

Another important requirement is that the peptide controls must be stable. This includes stability over time as well as stability after treatment with organic solvents (alcohol, xylene) or heat. Without stability, inter- or intra-laboratory variability could instead be ascribed to differences in the peptide controls length of storage or susceptibility to treatment conditions. Instability could obscure differences in the analytical component of immunohistochemical staining. 

Figure 7.6 Peptides do not denature after baking (dry heat) or deparaffinization and antigen retrieval (wet heat). Peptide-coupled slides were treated as indicated on the Y-axis and then immunohistochemically stained. In this particular example, an ER peptide with an ER MAb was used. The resulting peptide spot intensity (mean pixel intensity on a 1-256 scale) was measured and is shown on the y-axis.The data represent the means and SD or triplicate measurements. The experiments on the left (solid bars) and the right (hatched bars) were conducted at different times and have no connection to one another. Adapted with permission from Sompuram et al.6...

Zeheb R. Automating immunohistochemistry. In Immunohistochemical Staining Methods, 4th edition, ed. ME Key, pp. 103-106. Carpinteria, CA Dako, 2006. 

Immunohistochemical staining is very different from routine chromogenic stains such as H E. To utilize IHC stains for quantitative purposes, the user must understand these differences and perform the stain in both a standardized and repeatable manner. As has been described, image analysis... 

In 1991 Shi et al. published their seminal observation that high-temperature incubation of formalin-fixed, paraffin-embedded (FFPE) tissue sections in buffers for short periods led to improved immunohistochemical staining.1 However, more than 15 years later, heat-induced antigen retrieval (AR) remains largely an empirical procedure, requiring the optimization of several critical parameters by trial and error.2,3 Further improvements in AR will require an in-depth understanding of the chemistry of formaldehyde fixation and the molecular mechanism(s) underlying the AR method. 

Figure 16.8 Intensity of immunohistochemical staining as a function of the length of antigen retrieval time. All values represent the mean of triplicate measurements. The staining intensity of a peptide array that was not formalin fixed is shown at the far right of the graph, as a control. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

Shin HJ, Shin DM, Shah T, et al. Methods in pathology. Optimization of proliferating cell nuclear antigen immunohistochemical staining by microwave heating in zinc sulfate solution. Mod. Pathol. 1994 7 242-248. 

A thin, rather uniform layer of cells, well preserved is prepared on glass slide for immunohistochemical staining. 

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). (See text for full caption).

Commercially available P-gal usually is isolated from Escherichia coli and has a pH optimum at 1-1.5. By contrast, mammalian p-galactosidases usually have a pH optimum within the range of 5.5-6 thus, interference from endogenous P-gal during immunohistochemical staining can be avoided. 

Due to the relatively high-molecular-weight of the enzyme, conjugates formed with antibodies and P-gal tend to be much bulkier than those associated with AP or horseradish peroxidase. For this reason, antibody conjugates made with P-gal may have more difficulty penetrating tissue structures during immunohistochemical staining techniques than those made with the other enzymes. 

Circle sections with a hydrophobic barrier pen (e.g., Dako Pen, S2002) and proceed with immunohistochemical staining protocol. Do not allow sections to dry for the remaining procedure. 

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