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Figure 2. The staining of lipid A, the core component of LPS, utilized the lipid A binding moieties LALF (A, D, G) and biodipy-conjugated polymyxin B (E, F, I). For all micrographs, the specimens were fixed in freshly prepared 4% paraformaldehyde in PBS (10 min, room T), then blocked successively with 0.1 M glycine in Tris-buffered saline (TBS) and with 5 mg/ml BSA in the same buffer. Specimens were examined with Zeiss Axioplan and Leica DMIRBE confocal microscopes. To reduce the likelihood of contamination with exogenous bacterial LPS, all reagents were prepared in pyrogen-free water (Sigma cat W3500); glassware and microscope slides (Fisher Superfrost/Plus cat 12–550-15) were treated at 180°C for 4 h to remove LPS. Staining is seen with our positive control, the Gram-negative bacterium, E. coli (A), the green alga Chlorella sp., strain NC64A (D), the endosymbiotic algae of the cilliate protozoan, Paramecium busaria (E), the intracellular algae of the green hydra, Chlorohydra viridissima (F), isolated chloroplasts from the garden pea (G), and chloroplasts in paraffin sections of pea leaf tissue (I). The intracellular algae (E, F) and chloroplasts in situ (I) stained more intensely than the organelles and other membrane systems of the host cells. When LALF was omitted from the staining regimen (B, C-fluorescence and phase contrast micrographs of a clump of E. coli exposed to all reagents for LALF immunostaining but with the omission of LALF; H: fluorescence micrograph of isolated pea seedling chloroplasts stained in a manner identical to the bacteria of panel B) or when polymyxin B was preincubated with bacterial LPS (data not shown), staining failed to occur. The pairs of photomicrographs (panels A, B and G, H) were photographed and processed identically. The brighter background fluorescence in panel A is due to staining of LPS that has adsorbed onto the glass microscope slide from the buffer in which the bacteria were suspended and presumably is material that had been shed into the buffer by the bacteria. The autofluorescence of chlorophyl did not contribute to the fluorescent signal from algae (not shown) or chloroplasts (H). Bar, 10 µm.
