Secondary detection

As a test of reproducibility of spot printing and staining, a 10 spot by 10 row array (100 spots total) were printed on three slides. All slides were stained in a single staining run, using the same secondary detection chemistry with DAB as a chromogen. While DAB cannot provide accurate photometric measurements, it can provide relative comparisons from one spot to another on the same slide. When the stained control slides were analyzed in this... 

No. Technique name Probing radiation Main nuclear reaction Detected radiation Sources Primary/secondary detected elements... 

The current second-generation tools offer more promise due to (a) a focus on secondary detection techniques for peak tracking and (b) better automation tools offered by instrument vendors. 

The basic tool for detecting antibodies binding to a specific antigen is the ELISA, or Enzyme Linked Immuno-Sorbent Assay [3]. In this assay, antigen-specific antibodies are detected by antigen-mediated attachment to a solid support and secondary detection with an Fc-specific enzyme-labeled secondary antibody (Fig. 2). For hapten immunization, ELISA is performed using a carrier protein, typically BSA (bovine serum albumin), different from the carrier protein used for immunization. In this manner only antibodies with binding specificity to the hapten are revealed by the assay. 

In tyramide signal amplification (TSA), the target cells are labelled with an antibody or a nucleic acid probe, followed by secondary detection with a horseradish peroxidase (HRP) labelled antibody. HRP activates multiple copies of fluores-cently labelled tyramide derivatives, yielding fluorescent tyramide radicals that are deposited in the vicinity of the HRP-target interaction site (Fig. 5). 

Recommendation 13 was made to increase secondary detection methods. It stated Buncefield-type sites should errtploy measures to detect hazardous levels of flamma-ble vapors in secondary containment. This evaluation should include consideration of the following ... 

It is advisable to match the donor species of the serum with that of the host of the secondary detection antibody. During the longer steps where the antibody is incubated with the CNS tissue, it is advisable to add 1 mM sodium azide to the blocking solution to prevent bacterial growth. 

The general strategy for FISH is to equilibrate the tissue in buffered formamide, to add the probe(s), and to denature both the chromosomal DNA and probe together by heat treatment. The probe is then allowed to anneal for several hours at an appropriate temperature, unbound probe is washed away, and secondary detection (if required) is performed. The nuclear DNA is then usually counterstained, and the sample is mounted for microscopy. For experiments in which immunolocalization of other cellular components is desired, these staining steps can conveniently be performed after hybridization. 

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