Immunohistochemical staining procedure

Transfer slides to post-HIER immunostaining buffer, and continue with the immunohistochemical staining procedure (see Notes 10 and 12-16). 

Of the total number of commercial diagnostic assays utilizing antibody—enzyme conjugates, GO is employed in less than 1% of clinical tests. The enzyme remains, however, an important tool in many assays developed for research use. One particular advantage to the enzyme is that there is no endogenous GO activity in mammalian tissues, making it an excellent choice for immunohistochemical staining procedures. 

Mild to moderate hemolysis in antiserum resulting from sub-optimal bleeding techniques probably does not interfere with most immunohistochemical staining procedures, but excessive hemolysis should be avoided. If excessive hemolysis or lipemia is encountered, isolation of the immunoglobulin fraction from the antiserum may be necessary. Such isolates will usually appear colorless and clear. Discard all immunochemicals, including antisera and normal non-immune sera contaminated with bacterial growth. Their use in... 

Place slides on a Dako Autostainer instrument and proceed with staining. The sections should not dry out during the treatment or during the immunohistochemical staining procedure. 

Because of these limitations, polymer-based immunohistochemical methods that do not rely on biotin have been introduced and are gaining popularity (5). These methods utilize a unique technology based on a polymer backbone to which multiple antibodies and enzyme molecules are conjugated. In the EPOS (Enhanced Polymer One Step) system, as many as 70 enzyme molecules and about 10 primary antibodies were conjugated to a dextran backbone. This allowed the entire immunohistochemical staining procedure, from primary antibody to enzyme, to be accomplished in a single step (6). On the other hand, one limitation of this method was its restriction to a select group of primary antibodies provided by the manufacturer, and not suitable for user-supplied primary antibodies. 

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. 

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. 

Ki-67 antigen immunohistochemical staining is a simple and reliable procedure for studying tumor proliferative activity in frozen or formalin-fixed, paraffin-embedded tissues, including archival specimens. This antigen can be retrieved on sections of formalin-fixed and paraffin-embedded tissues by autoclave treatment (Fig. 10.1) or microwave heating (Fig. 10.2). Both methods are reliable and are presented later. 

Almost all pre-embedding staining procedures consist in the following steps (i) prefixation to obtain maximum permeabilization of the tissue with the best acceptable structural preservation and minimal loss of antigens (ii) immunohistochemical incubations (iii) further fixation (iv) revelation of the enzyme (v) postfixation (e.g., with osmic acid to render the DAB product electron dense) and, (vi) dehydration and embedding. Extensive washings are included between the various steps. The enzyme-generated product should be insoluble, both in water and alcohol. 

Detection of metastatic disease may occasionally require the use of immunohistochemical stains in challenging specimens such as postradiation lymph nodes. Subtle post-treatment residual tumors can be detected clinically using PET/CT scans, but histologically these isolated foci may show only granulomatous or necrotic tissue without viable tumor. In these cases, cytokeratin stains can be helpful to identify the etiology of the necrotic deposits. Sentinel lymph node examinations are occasionally used in head and neck surgery. Currently no standard practice for using immunohistochemistry to examine the sentinel lymph nodes exists, and indeed the procedure is fairly unreliable for mucosally based tumors. 

Currently, biopsies of the prostatic urethra are recommended as a staging procedure in patients undergoing intravesical treatment for superficial bladder tumors. If intraductal urothelial carcinoma is identified on TURP or transurethral biopsy, patients will usually be recommended for radical cystoprostatectomy. The finding of intraductal urothelial carcinoma has also been demonstrated to increase the risk of urethral recurrence following cystoprostatectomy, such that its identification may also result in prophylactic total urethrectomy. Immunohistochemical stains for basal cells (high-molecular-weight cytokeratin, p63) may in some cases only outline the prostatic basal cells and in other cases label the intraductal urothelial carcinoma. 

This section addresses some of the most common problems encountered in immunoperoxidase procedures and the appropriate solutions to correct them. Note that we address manual immunohistochemical staining methods only see appropriate operator s manual for troubleshooting of automated immunostainers. 

Immunohistochemical Procedures for Light Microscopy The immunohistochemical staining using a biotin-streptavidin-peroxidase complex was performed according to the... 

Microspreading is the easiest and most rapid method for SC observation in 2 hr the material can be examined in the EM. After microspreading, any of the staining procedures and most of the immunohistochemical tests, as well as FISH, can be performed. Kinetochores and RNs are well preserved after PTA staining, which also shows all the SC components (Fig. 2). The main limitations of this procedure are the low efficiency with respect to the number of observed nuclei and the stretching of the SCs introduced by the technique. 

Incubate the sections in the secondary antibody conjugated to the desired fluorochrome (see Note 47) and proceed with standard immunohistochemical procedures. Fig. 2B demonstrates the appearance of an immunohistochemically stained lacZ-positive cell. 

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