Immunocytochemistry/ Immunofluorescence abreview for Anti-E. coli LPS antibody [2D7/1]

Good
Abreviews
Application
Immunocytochemistry/ Immunofluorescence
Sample
Drosophila melanogaster Cell (hemocyte with engulfed E. coli (DH5a))
Permeabilization
Yes - 0.1% Triton
Specification
hemocyte with engulfed E. coli (DH5a)
Blocking step
Serum as blocking agent for 15 minute(s) · Concentration: 5% · Temperature: 22°C
Fixative
Paraformaldehyde

Other product details

Incubation time
1 hour(s) and 0 minute(s) · Temperature: 22°C · Diluent: 5% normal goat serum 0.1% Tween
Dilution
1/200

Secondary antibody

Dilution
1/1000
Name
Non-Abcam antibody was used: goat anti-mouse Alexa 594
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor­ 594

Additional data

Additional Notes
Drosophila larvae were injected with DH5alpha bacteria, then hemocytes bled out 45 mins later for fixing, IFC. We were looking for a reagent that would stain/localize engulfed (phagocytosed) bacteria. We found that the Ab worked well in that it DID stain many engulfed bacteria well. We had another reagent that localized to phagosomes, and we could see, however, that not all of the phagosomes stained positive with this anti-LPS antibody. This could be because the bacteria were already degraded, OR because they were not sufficiently degraded. The latter may sound counter-intuitive however, it is the theory we favor for 2 reasons: (i) 45 mins is a short time window, and bacteria are not yet highly degraded yet and (ii) non-internalized bacteria present on the slide were NOT stained with the antibody (see picture). If the ligand is truly LPS, this would not make sense. However, the ligand has not been well-characterized, and we suspect it may be a bacterial molecule that is not exposed in Gram-negative bacteria until partial degradation in the phagosome.

Catherine Brennan

Verified customer

Submitted Jan 10 2018

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