Sequential staining

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-... 

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

Thomas ML, Szeto VR, Gan CM, Poise KA. Sequential staining the effects of sodium fluorescein, osmolarity, and pH on human corneal epithelium. Optom Vis Sci 1997 74 207-210. 

For electron microscopy, the preparations were fixed in 1% glutaraldehyde and 1% OSO4, and post-fixed in 1% OSO4. The fixed preparations were dehydrated through a graded ethanol series and were embedded in Epon 812 resin. The sections were sequentially stained with 4% uranyl acetate and 0.4% lead citrate and viewed under the electron microscope. 

Volpi, N. Maccari, F. Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All. Electrophoresis 2002, 23, 4060-4066. 

Is there any other approach or concept that can directly measure protein amount in the tissue section Ten years ago, Roth et al.38 documented a novel method, named the Midwestern assay. This method is based on using two chromogens, soluble and insoluble, for the IHC staining process, to produce sequential production of soluble and insoluble reaction products. The soluble IHC product is used to measure the amount of antigen (protein) by spectrophotometry, while insoluble product indicates the localization of protein in the tissue section. Their experimental results demonstrated that soluble reac-... 

Van der Loos CM, Teeling P. A generally applicable sequential alkaline phosphatase immunohistochemical double staining. I. Histotechnol. 2008 31 119-127. 

A collection of useful protocols for the simultaneous immunoenzyme staining of two or more tissue antigens is available on the websites http //www.protocol-online.org/prot/Immunology/ http //www.vectorlabs.com/Protocols/MLB.pdf and http //www.ihcworld.com/. The protocols given in this chapter are practiced in the authors laboratory. These methods fall into two main categories (a) simultaneous immunoenzymatic double staining, and (b) sequential immunoenzymatic double/ multiple staining. 

Basic protocol for sequential immunoenzymatic double staining (Adapted from http //www.vectorlabs.com/ and http //www.ihcworld.com/ books/Dako Handbook.pdf)... 

Stripping Buffers for Sequential Immunoenzymatic Double Staining... 

Use the relevant visualization steps described in Subheading 3.3. for IGSS, IPO, or lAP, in sequential reactions. Sections stained for immunofluorescence should be mounted with a glycerol-gel solution containing an antifading agent such as Vectashield (Vector). 

To assess any antibody adsorption to Leica manufactured vials, a primary antibody was stored in the reagent vials for up to 1 mo at 4°C. Subsequently, the antibody was removed, and the remaining reagents of an immunostaining run were sequentially added, as in the Cadenza vial and Code-On isolon experiments. Slight vial staining was seen after the 1-mo incubation period, indicating minimal antibody adsorption. 

To assess possible adsorption of antisera to the Ventana dispensers, a Ventana pipeter filled with L26 antisera was stored at 4°C for up to 1 mo. At the end of the storage period, the antisera was decanted, and the pipeter thoroughly rinsed with PBS. The pipeter was then stained to assess antibody adsorption using sequential application of standard immunostaining reagents. After 4 wk of storage, there was no detectable immunoreactivity in the Ventana pipeter. 

Figure 1. Morphology of sequential IPNs. (a) Crois-poly (ethyl acrylate)-m/er-crojs-polystyrene, showing typical cellular structure and a fine structure within the cell walls, (b) Cross-poly (ethyl acrylate)-/ /cr-cross-polystyrene-s/a/-(methyl methacrylate), showing smaller domain structure. PEA structure stained with OsO. (Reproduced from ref. 5. Copyright 1972 American Chemical Society.)...

Figure 2. Morphology of various cross-polybutadiene-in/er-cross-polystyrene sequential IPNs and graft copolymers via transmission electron microscopy. The double bonds in the polybutadiene phase are stained dark with osmium tetroxide. (Reproduced from ref. 15. Copyright 1976 American Chemical Society.)...

Because the ability to dye one protein may differ from that of another in an electropherogram, an unknown protein mix should become stained by different methods. Some of the procedures can be done sequential in the same gel, e.g., additional Coomassie staining after silver staining. 

Fig. 11 Visualization of the sequential progress of branching stage by staining with rose bengal. DC-derivatized PST surface (GO) was graft-copolymerized with CMS and dimethylaminoethyl acrylamide (DMAEMA), and subsequent quarternization, while narrowing the irradiation area in each polymerization stage (item, GI item, GI+GII item, GI+GII- -GIII) by the combination of three kinds of masks with linear openings (line widths 2 mm for GI, 1 mm for GII, 0.5 mm for GUI). Bar=0.5 mm...

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