In this study, we are reporting a different DNA immobilisation approach using the latex-gold nanoparticles as the DNA probe immobilisation substrate to develop a highly sensitive detection system for V. cholerae DNA.
We then used this substrate to develop highly sensitive HTS assays (Z'>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases.
DAB (3,3′-diaminobenzidine solution; SIGMA, fast DAB) was used as a substrate to develop specific staining.
After washing, TMB was used as substrate to develop color and plates were read at 450 nm on a SpectraMax M4 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
A biotin-labeled goat anti-mouse IgG secondary antibody was used with horseradish peroxidase-conjugated streptavidin (Vectastain ABC; Vector Laboratories, Burlingame, CA, USA), and 3,3 -diaminobenzidine substrate to develop a color reaction.
After a series of PBS washes, slides were covered with peroxidase substrate to develop color and then washed in three changes of dH2O and counterstained in 0.5% (w/v) methyl green for 10 minutes at room temperature.
PrPSc immunostaining was performed as described above with the exception that Powervision AP (ImmunoVision technologies, Brisbane, CA, USA) was used as secondary antibody and 5-bromo-4-chloro-3-indoxyl phosphate and nitro blue tetrazolium chloride (BCIP/NBT DakoCytomation, Glostrup, Denmark) were used as substrate to develop an insoluble blue reaction product.
Samples were then washed several times in PBS, the slides were covered with peroxidase substrate to develop color and then wash in three changes of distilled H2O and counterstained in 0.5% (vol/vol) methyl green for 10 minutes at RT. Cell apoptosis was measured by counting under a light microscope the number TUNEL positive cells, which represent apoptotic cells and possibly some necrotic cells.
Finally, the slides were covered with the mixed solution of DAB and HRP substrate buffer to develop the color and washed with deionized water, and followed by hematoxylin counterstaining.
The sections were finally incubated in peroxidase substrate solution to develop color, followed by washing in water, counter staining with hematoxylin (Vector Laboratories), clearing in xylene and cover-slipping with Permount (Fisher Scientific, Pittsburgh, PA, USA).
