Fifteen minutes after injection, the embryos were fixed, and the localisation of the bacteria was checked by using transmission electron microscopy (TEM).
We identified the apico-basal localisation of representative lines in fixed tissues by comparing it with the localisation of a phosphotyrosine epitope (p-Tyr), which is enriched at adherens junctions (Fig. 4) (Muller and Wieschaus, 1996).
To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/methanol (40∶60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1∶1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France).
For immunofluorescence detection, cells were fixed in 3% paraformaldehyde (15 min) and localisation of proteins was detected as previously described [ 7].
Because the initial response was not clear in all subjects, the time window for source localisation was fixed at 27 44 ms, which was the mean ± SD of the initial response in those subjects and for all conditions where it was clearly expressed.
To check the localisation of the tagged SOD A, epimastigotes were fixed in 4% paraformaldehyde and dried onto slides.
The localisation of this complex was then examined directly on fixed, permeabilized cells.
Cells were viewed live to map intracellular drug localisation by detecting their natural fluorescence or they were fixed for Pgp immunofluorescence.
The very fact that Pol II moves across genes during elongation complicates studies of its localisation in comparison to activities which are recruited to fixed loci such as promoters.
Cultures were grown for seven days, and then gels were fixed and whole gel immunofluorescence carried out to assess co-localisation and polarisation.
To assess the pathway of STS-induced apoptosis, and how it is sensitized in PINK1 kd cells, we used 0.75 µM STS for 4hrs, fixed cells at different intervals and performed double immunofluorescence to assess the localisation of Bax and cytochrome c.
