Slides for Immunohistochemical Staining

In order to prevent detaching of tissue sections during immunohistochemical staining and unmasking, specially coated sides (with silane or poly-L-lysine) or electrostatically treated sides (commercially available) must be used. The following simple protocol provides suitable slides for immunohistochemical staining and in situ hybridization. 

Incubate slides in 10% silane (3-aminopropyltriethoxysilane)-acetone solution for 1 min (20 ml stock silane solution mixed with 180 ml acetone) 

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

Prior to immunohistochemical staining, paraffin sections must be properly mounted onto slides, and then deparaffinized and rehydrated. To help adherence to the glass and decrease the chances of sections dissociating from the slides, paraffin tissue sections should be mounted on tissue-adhesive-coated slides. The use of tissue-adhesive-coated slides is especially important for paraffin tissue sections undergoing heat-induced antigen retrieval (see Sect. 6.1.1). 

As for paraffin sections, it is advisable to mount cryosections also onto adhesive-coated slides in order to decrease the chances of sections dissociating from the slides in the course of immunohistochemical staining. Once mounted on slides, cryosections are air-dried and fixed, usually in acetone or methanol. Aldehyde... 

Transfer slides into post-HIER blocking buffer for 15-20 min (see Notes 10-12) and continue with the immunohistochemical staining procedure (see Notes 13-17 and Chapters 25, 26, 28, and 29). 

Fig. 6. In situ conversion reaction in brain slices on glass slides (Bessen et al., 1997). A distinct autoradiographic image is seen with scrapie-infected, but not uninfected control, brains. If the brain slices are solubilized after the conversion reaction and analyzed by SDS-PAGE/autoradiography, then PK-resistant S-PrP conversion products similar to those produced in cell-free conversion reactions (see Fig. 5) are observed. Higher magnification images (bottom panels) show that the pattern of in situ conversion product closely matches that of immunohistochemical staining for PrP-res in regions known to contain either amyloid plaques or diffuse apparently nonamyloid deposits.

Pathiam Hardware -independent,Web-enabled software for viewing and analyzing immunohistochemically stained slides. Pathologists can automatically generate reports containing a complete quantitative analysis of each sample. www.bioimagene.com... 

Rat Tissues Immunohistochemical staining was carried out as described previously (14). Briefly, sections were treated with RNase A(100 ug/ml) at 37°C for 2 hr and then with 2.5N HCl at room temperature for 45 min. Slides were incubated with 5 % normal goat serum for 20 min and then with anti-DMAB-DNA antibody for 60 min at 37 C. After treatment with 0.3 % H2O0 for 30 min at 37°C, binding of the antibody was visualized by the ABC method(37°C for 30 min) with 0.05 % diaminobenzidine (for 10 min). The sections were also weakly counterstained with hematoxylin before dehydration through graded ethanols and xylene, and mounting. 

Figure 20.1 shows a cerebral tumor from the lumbosacral region of a middle-aged woman. The H E-stained slide reveals a neoplasm with mainly epithelioid cells and a few clear cells, and abundant round to oval nuclei with fine chromatin (Table 20.6 see Fig. 20.lA). Its IHC is focally positive to epithelial membrane antigen (EMA) (see Fig. 20.IB). Boxes 20.1 and 20.2 show carcinoma, chordoma, craniopharyngioma, pituitary adenoma, and meningioma to be EMA positive. This tumor is negative for cytokeratin (CK) CAM5.2 (see Fig. 20.1C), so it does not fit the immunohistochemical...

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