Biology Reference
In-Depth Information
3. Lids from 24-well tissue-culture cluster plates. These do not need to be sterile
and can be saved after use in other experiments.
4. Phosphate-buffered saline (PBS): 137 m
M
NaCl, 2.68 m
M
KCl, 8.1 m
M
Na
2
HPO
4
, and 1.47 m
M
KH
2
PO
4
(pH 7.4) in tissue-culture grade deionized water.
Also, PBS supplemented with 10 mM glucose. Sterile filter (0.2 μm) and store at
room temperature and 4 °C.
5. “PAB” washing/blocking buffer: PBS supplemented with 0.5 mg/mL NaN
3
and
5 mg/mL bovine serum albumin. Sterile filter and store at 4 °C.
6. Anti-
H. pylori
antibody (Ab): Rabbit anti-
H. pylori
polyclonal Ab (#YVS6601)
from Accurate Chemical and Scientific Corp. (Westbury, NY) (
see
Note 2
).
7. Secondary Ab: Fluorescein isothiocyanate (FITC)- and rhodamine-conjugated
F(ab´)
2
secondary Abs (Jackson ImmunoResearch Laboratories, West Grove, PA).
Rehydrate with sterile deionized water according to the manufacture's directions
and store at 4 °C.
8. Gelvatol mounting medium (
see
Note 3
): Mix 2.4 g of polyvinyl alcohol with 6 g
of glycerol, 6 mL of deionized water, and 12 mL of 200 m
M
Tris-HCl (pH 8.0)
in a beaker on a stir plate for several hours until dissolved. Transfer the solution
into a 50-mL polypropylene tube, heat the solution to 50 °C for 10 min, and then
clarify by centrifugation at 5000 ×
g
for 15 min. Decant the supernatant into a new
50-mL polypropylene tube and add 625 mg of 1,4-diazobicyclo-[2.2.2]-octane
(DABCO). Invert the tube to dissolve DABCO. Store 1 mL aliquots of mounting
medium in a frost-free freezer at -20 or -80 °C. DABCO is essential as it reduces
photobleaching of fluorescence.
9. Fluorescence microscope: Upright fluorescence microscope with 40×, 63×, and
100× phase contrast objectives and filters for detection of both FITC and
rhodamine fluorescence.
3. Methods
3.1. Preparation of Acid-Washed Coverslips
Acid washing removes manufacturing residue, oils, and other contaminants
such as lipopolysaccharide (LPS) that may inadvertently activate macrophages
or cause high non-specific background fluorescence. Transfer one package
(1 ounce) of 12-mm round glass coverslips into a small glass bottle. Pour nitric
acid over the coverslips making sure they are all submerged. Cap the bottle
and incubate in the fume hood for at least 48 h. Carefully decant the acid and
then rinse the coverslips with 15-20 changes of sterile deionized tissue-culture
grade water (pour water over the coverslips, cap bottle, rotate gently, decant
water, and repeat). Rinse the coverslips with two changes of 95% ethanol to
remove residual water. Store coverslips in 70% ethanol at room temperature.
Use after
≥
16 h in ethanol.
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