Biology Reference
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5. Dulbecco's phosphate-buffered saline (D-PBS) with or without MgCl 2 and CaCl 2
(Invitrogen).
6. D-PBS saline without MgCl 2 and CaCl 2 , supplemented with 1g/L d-glucose:
dissolve 2.5 g of d-glucose in 10 mL of D-PBS and add it to 500 mL of D-PBS
by filtering it through a 0.2-μm filter in a tissue culture cabinet.
7. L-929 mouse fibroblasts (ATCC No CCL-1) grown in DMEM supplemented
with 10% FBS and 2 m M l-glutamine.
2.3. Immunofluorescence Microscopy
1. 12-mm round 0.1-mm thick glass coverslips: degrease coverslips in 100%
acetone for 5 min, rinse them five times in 70% ethanol, dry them on Whatman
paper, and sterilize them by autoclaving between Whatman paper discs in a
glass Petri dish.
2. High-Precision DUMONT tweezers #5 and #7 (Ted Pella Inc., Redding, CA).
3. PBS: Prepare 10× stock with 1.37 M NaCl, 27 m M KCl, 100 m M Na 2 HPO 4 ,
and 18 m M KH 2 PO 4 (adjust to pH 7.4 with HCl if necessary). Autoclave before
storage at room temperature. Prepare working solution by diluting one part 10×
stock with nine parts of water ( see Note 1 ).
4. Paraformaldehyde (PFA; Sigma, P6148): Prepare a 25% stock by dissolving
10 g of PFA in 30 mL of water and heat at 70 °C on a stirring hot plate in a
fume hood. Add drops of 10 N NaOH until PFA completely dissolves. Aliquot
in Eppendorf tubes and store at -20 °C. A 2.5% working solution is prepared
by thawing a 25% stock aliquot at 70 °C and diluting it in 9 mL of water. pH is
adjusted to 7.2-7.4 with 1 N HCl (
15 μL) using a pH strip.
5. Quenching buffer: 50 m M NH 4 Cl in PBS.
6. Permeabilization/blocking buffer: 10% horse serum (Sigma) and 0.1% (v/v)
saponin in PBS.
7. Antibody dilution buffer: 10% horse serum (Sigma) and 0.1% (v/v) saponin in
PBS.
8. Primary antibodies: mouse monoclonal anti- F. tularensis LPS (U.S. Biological,
Swampscott, MA), rat monoclonal anti-mouse LAMP1 (1D4B; developed by
J. T. August and obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the NICHD and maintained by The University of
Iowa, Department of Biological Sciences, Iowa City, IA), rabbit polyclonal anti-
calnexin (SPA-860; Stressgen Bioreagents, Ann Arbor, MI), rabbit polyclonal
anti-calreticulin (PA3-900; Affinity BioReagents, Golden, CO), mouse anti-
PDI, (SPA-891; Stressgen Bioreagents), goat polyclonal anti-EEA1(N-19; Santa
Cruz Biotechnology, Santa Cruz, CA), rabbit anti--COP (PA1-061; Affinity
BioReagents), and mouse anti-GM130 (BD Transduction Laboratories, San Jose,
CA).
9. Secondary antibodies: Alexa Fluor™488-conjugated, Alexa Fluor™ 568-
conjugated (Molecular Probes, Carlsbad, CA), and cyanin-5-conjugated (Jackson
ImmunoResearch Laboratories, West Grove, PA) donkey anti-mouse, anti-rat,
anti-rabbit, and anti-goat antibodies.
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