Streptavidin coated slides

Place the slide in a 50 ml Falcon tube and centrifuge at 1,000 x for 5 min at 20°C to spin dry. Streptavidin-coated slides were placed into slide boxes, sealed in Ziploc bags and stored at -20°C. 

Load the streptavidin-coated slides onto the print bed of the QArray II. 

To make a reference for the 3-D NPH-streptavidin (3-D NPH-SA), the solution including no poly (NAM-co-NAS) was dispensed on the Au-coated slide activated with succinimide esters. The product was referred to as 2-D reference (2-D SA). 

The spotting experiment must be finished within 1 h after preparing streptavidin/poly (NAM-co-NAS) mixed solutions. Because the reaction between the succinimide esters of poly(NAM-co-NAS) and the amino groups of SA molecules and hydrolysis of the succinimide esters of poly (NAM-co-NAS) proceed before printing on substrates in 384-well, 3-D NPH-SA is difficult to be made on Au-coated slide activated with succinimide esters. 

Sections of formalin-fixed and paraffin-embedded tissues are placed onto silane-coated slides, deparaffinized, and rehydrated. They are placed in 0.1 M EDTA (pH 8.0) and exposed to steam heat for 30 min. The slides are cooled for 5 min, rinsed in tap water, loaded onto the ES Autostainer, and treated with Protease 2 (Ventana) for 8 min. Incubation is carried out in the primary 34 3E12 antikeratin (diluted 1 10 in PBS) for 32 min and in biotinylated secondary antibodies followed by streptavidin for 8 min each. The staining is visualized on the instrument using 3-amino-9-ethylcarbazole. Between each step, the slides are rinsed in Tris-buffered saline. The results of this protocol are shown in Figure 8.8. 

In general, any microarray printer could be used to print the arrays and the printing procedures described below can typically be carried out at room temperature, preferably with the atmosphere in the print chamber humidified to ca. 50% to reduce evaporation rates. If necessary, either the print bed or the entire the printing device could be cooled to 4°C however, we have typically found that this provides little benefit in terms of protein activity on the resultant arrays. Here we describe one specific set of parameters which work well on streptavidin-coated glass microarray slides at room temperature using a Genetix QArray II robot equipped with 16 x 300 mm tipped solid pins (Figs. 6 and 7 see Notes 7-12) ... 

Fig. 6. Printing onto a streptavidin-coated glass microscope slide using 16 solid pins.

Photo-polymerization was conducted at 254 nm under a modified fluorescent microscope. Allyl-oligonucleotides were employed with methylene blue as the free radical initiator for crosslinking. In the case of proteins, acryloyl streptavidin was first immobilized so that biotinylated proteins could be applied to the gel pad (Vasiliskov et al., 1999). One of the major drawbacks with the gel pad approach was that separate pads had to be manufactured instead of coating the enhre slide with the gel and then printing down the microarray. 

Fig.26 Pseudo-color time-resolved image of polystyrene nanobeads containing Pt porphyrin and conjugated to streptavidin immobilized to a biotinylated microarray surface (black teflon coated 96-well glass slide, spot diameter 1 mm, Erie Scientific) in different concentrations (25, 15, 10, 5, Ong streptavidin per well) [167]...

Fig. 3. Schematic of staining process of SARS-CoV immunochip. (1) Spotting A high-precision robot transfers the samples, SARS-CoV proteins, and glycans of various complexities, from 96-well plate to nitrocellulose-coated glass slides. (2) Staining Before staining, the slides are rinsed with IX phosphate-buffered saline (PBS), and blocked with 1% bovine serum albumin (BSA)-PBS containing 0.05% NaN3 and 0.05% Tween-20. They are subsequently incubated with horse anti-SARS sera. The primary antibodies captured by microarrays are detected using biotinated anti-horse immunoglobulin (Ig)G, and visualized by Cy3-streptavidin.

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