Streptavidin Alexa

Slices were rinsed in PBS three times and permeabilized in PBS containing 0.3 % Triton X-100 for 4 h in room temperature. The slices were then incubated for another 4 h in 2—4 pg/mL streptavidin Alexa 568 in PBS containing 0.3 % Triton X-100. Slices were thoroughly washed in PBS at least three times and then mounted on glass slides, air-dried, and coverslipped using VECTASHIELD mounting medium. 

Standard immunohistochemistry was carried out on sections prepared as described (Sect. 3.1). They were rinsed in PBS (2x10 min) and then placed in antibody birffer for 30 min at 18-20 °C. Sections were then incubated in the same solution containing the primary antibody (250 pL/well in a 24-well plate, three sections/well) for 70 h at 4 °C (control sections incubated in antibody buffer only). Sections were then washed three times for 10 min each in PBS at room temperature before incubation with secondary antibody diluted in the antibody buffer (including controls) for 3 h at 18-20 °C. Sections were washed twice for 10 min in PBS (three times for 10 min for CTIP2 detection). The sections used for CTIP2 detection were then incubated with streptavidin-Alexa 568 for 2 h at 18-20 °C diluted in antibody buffer, followed by two rinses in PBS. All sections were mounted on glass slides and coverslips applied with VECTASHIELD antifade solution. 

A surface coated with a SAM of 3-mercaptoproponic-acid, was used for the covalent attachment of Alexa-Fluor 647 labeled streptavidin (AF-SA) by means of NHS-active ester chemistry [38]. This reaction yielded an SPR minimum shift of A0 O.27° and contributed a fluorescence intensity of A/A 350 eps (layer b ). Subsequently, the alternate binding of biotinylated IgG and streptavidin from 20 pg ml-1 solutions was monitored. 

Figure 5.6 Odyssey Scanner results showing CCA enhancement of IRDye 800CW (Top two rows) and Alexa Fluor 680 (Bottom two rows) labelled streptavidin on glass slides. Scanner image (top) shows four CCA preparations (A-D) used to enhance the two near-IR fluorophores, as well as the fluorophores spotted on plain glass without CCA nanostructures added (Dye Alone). Bar graph (bottom) shows the relative fold enhancement over the Dye Alone samples for each of the CCA preparations. Error bars are shown that reflect the deviation between two samples for each preparation.

Streptavidin-fluorochrome conjugates provide higher sensitivity than two-step indirect techniques. The use of Alexa dyes instead of the traditional fluorochromes enhances brightness and detection sensitivity of immunofluorescence methods several-fold (12). 

While no QDs have been approved for clinical use to date, they have proven useful in both in vitro and in vivo diagnostics in animal studies. In vitro, QDs have begun to replace traditional fluorescent dyes due to the advantages discussed above. Wu a demonstrated this by the conjugation of immunoglobulin (IgG) and streptavidin to QDs with different emission spectra and showed how QDs can be used as a superior marker for both nuclear antigens and microtubules relative to Alexa 488 dye. 

Alexa 568 streptavidin conjugate (Invitrogen , Life Technologies, Grand Island, NY). 

Since the neurons targeted by whole-cell patch clamp recording were also filled with biocytin (0.2 %) we were also able to identify the shape and position of these P -GFP neurons within the cortical network. We used streptavidin conjugated to a red fluorescent probe (Alexa 568). Streptavidin reacts directly with biotin (biocytin) within the filled neuron, labeling only the filled neuron in the red fluorescence channel of the wide-field microscope. Typically we obtained recovery percentages of 80-100 % of neurons using the protocol below. 

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