Rate and Extent of Helicobacter pylori Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 145

Fig. 1. Set up for sample staining and washing. Materials for cell staining, washing,
and mounting coverslips are shown.

second stock of anti- H. pylori Ab. You will need 30 μL of Ab diluted in PAB for
each coverslip.
6. Using forceps, lift a coverslip off of the dish lid and drain Ab by touching the
edge of the coverslip to the pile of Kimwipes. Wash coverslips sequentially in
four microbeakers of PAB, draining thoroughly between each wash. Dry the
back of the coverslip and return it to the 24-well dish lid. Overlay with 30 μL
of anti- H. pylori pAb and incubate for 30-60 min. Because macrophages are
permeabilized, the anti- H. pylori Ab now has access to both intracellular and
extracellular bacteria.
7. Prepare FITC-conjugated secondary Ab diluted 1:200 in PAB. Wash coverslips
in microbeakers of PAB as described above and return to the dish lid. Overlay
with 30 μL of secondary Ab and incubate for 30-60 min.

3.6. Mounting Stained Coverslips onto Slides
Up to six coverslips will fit onto one slide. It is important to place the
coverslips as close as possible to the marking area of the slide ( see Fig. 1 ).
Samples mounted near the far right edge of the slide cannot be viewed because
this part of the slide rests on the microscope stage.
Thawone tube of mountingmedium.Workingwith coverslips for one slide at a
time, place six small drops ( 15 μL) of gelvatol mounting medium onto the glass

slide.Wash each coverslip sequentially in four beakers of PABas described above
followed by a final rinse in deionized water. The water rinse prevents a dried salt
crust from forming on mounted coverslips. Blot excess water off the back of the
coverslip and then invert it (cell side down) onto a bead of mountingmedium (start
from one edge of the coverslip and lower it slowly to avoid trapping air bubbles).
Repeat for the other coverslips on this slide. Gently press down on the top of each
coverslip to ensure that their edges are not overlapping (this will also push out
small air bubbles). Remove any excessmountingmediumusing an aspirator. Place
slides in a cardboard slide folder; this keeps the slides flat and protected from light.

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