Staining Protocols

Figure 7.5 Stability of peptide controls, for HER-2 (top panel), progesterone receptor (PR) 636 monoclonal antibody (middle panel), and estrogen receptor (ER) 1D5 monoclonal antibody (bottom panel). The dotted line represents the immunoreactivity of peptide controls stored at room temperature. The slight dip in immunoreactivity at the 3-month time interval is related to a slight inconsistency in the staining protocol (immu-nohistochemical detection) rather than an actual decrease in stability. Each time point is the mean + SD of four replicate peptide controls. Copied with permission from Bogen et al.9...

Performing appropriate antigen retrieval before initiating the staining protocol. 

Identification and tracking of individual slides and matching them to the proper staining protocol. 

Prevention of evaporation, which would cause the slides to dry out during the staining protocol. 

During the tissue fixation process, proteins are cross-linked, causing some epitopes to become undetectable by the staining protocols.10 HIAR reverses this effect, allowing these epitopes to be stained, and therefore has become increasingly important for many IHC staining protocols.19-22 However, the available automated IHC platforms vary in their ability to perform online HIAR. 

For systems that do not offer online HIAR, these procedures must be performed manually or off-line prior to loading slides on the instrument. Online HIAR methods are usually found as part of the closed-type automated staining systems, and are therefore less flexible in terms of what a technician can do to change the HIAR portion of a staining protocol to help optimize staining for particularly difficult antibodies. In spite of this, the ability to perform online HIAR is advantageous for many antibodies because it is extremely consistent and frees up technician time to complete other laboratory tasks. 

IHC stains as used for brightfield microscopy are sandwich procedures, consisting of sequential staining steps, each of which contributes amplification of the original signal. Specificity of the stain is provided by the primary antibody. The primary antibody also contributes to the sensitivity of the stain, but sensitivity is also the combination of each of the other steps of the stain protocol. It is critical that the steps used to amplify the signal in the stain process are not overdone, as these amplification steps can easily produce excess back-... 

The concept of a control is a device or mechanism to detect variations in the monitored procedure that are significant enough to produce a detectable result. In the case of IHC stains, the inability of subjective visual assessment to detect significant stain results has obscured this major source of variation for most users. This author has worked with quantitative evaluations of his-tochemical stains for many years. Initial attempts at quantitative evaluation of IHC stains clearly indicated that current practice does not provide adequate control of IHC stain protocols. 

The IHC stain procedure is a multistep staining protocol, the various steps intended to provide amplification of stain results. Therefore, a control system must include elements to control each step of the stain process. Such a control should also include a range of reactivities, and that range ideally would encompass the total expression range expected for the measured component. The control should also monitor each step of the multistep protocol. This author has devoted a number of years to this concept, resulting in a patented control for multistep staining processes.14 Such a control provides sufficient information to monitor every IHC stain run, and when the control is evaluated quantitatively, normalization of data from one stain run to another within the same laboratory, and even between laboratories. A process control is a measure of the stain protocol and does not take the place of a control for the primary antibody. While the primary antibody control should include range of expression level detection, a different primary control must be present for every primary antibody used in a stain run (Fig. 10.4). 

Floyd AD, Yaziji H. Process control for standardization of multiple step staining protocols. Arch. Pathol. Lab. Med. 2008 132 870-871. 

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. 

After deparaffinization and rehydration of sections, you may continue with the antigen retrieval if affordable (see Sect. 6.1.1) and the immunohistochemical staining protocol. 

If the staining protocol must be interrupted, sections may be kept in a buffer solution at 4°C until staining is resumed. 

Note Avoid exposing the cel Is to I ight as much as possible from step 15 onwards. For suspension cells carry out the staining protocol in an eppendorf tube till step 10. To adhere the cell... 

Alternate staining protocols provide for incubation of the tissue sections with the primary antibody at 4°C overnight. This sometimes results in enhanced staining withont an increase in backgronnd. 

Standardized staining protocols require standardized reagents to ensure reproducible results. Manufacturers of automated immunostaining instruments may supply proprietary buffers and wash solutions for their instrument, but the properties of these should be within the range of parameters that are used in manual staining. (The primary additives to manufacturer-supplied reagents are surfactants.)... 

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