Alexa fluor 647 conjugated goat anti mouse igm

The level of core 1 structure on cell surface was measured by flow cytometry with mouse mAb 3C9 (an in-house produced antibody) specific to core 1 glycosylation^{69 (link)}. Cells were incubated on ice with 3C9 mAb (undiluted hybridoma supernatant which is equivalent to 1:1 dilution) for 30 min, followed by washing and incubation with Alexa Fluor 647 conjugated goat anti-mouse IgM (1 µg/mL) (Invitrogen, catalogue: A21235) for 30 min. Diluting and washing was performed in PBS with 1% BSA and cells were resuspended for flow cytometry analysis (SONY SA3800). Mean fluorescent intensity of the binding of mAb 3C9 populations was quantified by FlowJo software (FlowJo LLC).

Primary Abs used in this study included biotinylated rat anti-mouse CD68 mAbs (clone FA-11, Bio-Rad), mouse anti-GFAP mAbs (clone G-A-5, MilliporeSigma), rabbit anti-GFAP (MilliporeSigma), mouse anti-MOG IgG mAbs (clone 8-18C5, gift of Claude Genain (University of California, San Francisco, California), rabbit polyclonal anti-human C5b-9 IgG (Calbiochem), mouse anti-plp1 IgG mAbs (clone plpc1, Thermo Fisher Scientific), rat anti-PLP1 AA3 IgG mAb hybridoma supernatant (61 (link)) targeting an intracellular C-terminus PLP1 epitope (produced in house), mouse O1 and O4 oligodendrocyte antigen hybridoma mAb (21 (link)) supernatants (produced in house) (IgM specific), and mouse anti-Flag IgG mAbs (clone M2, Stratagene). Isotype-matched fluorescent secondaries included Alex Fluor 488–conjugated donkey anti-human Fc fragment–specific IgG (Jackson ImmunoResearch), Alex Fluor 488–conjugated goat anti-human IgG (Invitrogen), Alexa Fluor 594–conjugated donkey anti-rat IgG (Jackson ImmunoResearch), Alexa Fluor 647–conjugated streptavidin (Invitrogen), Alexa Fluor 647–conjugated goat anti-mouse IgM (Invitrogen), Alexa Fluor 594–conjugated goat or donkey anti-mouse IgG (Invitrogen), HRP-conjugated goat anti-human IgG (Pierce Chemical Co.), and HRP-conjugated goat anti-rat IgG (Pierce Chemical Co). Secondary Abs were used at 1:800–1:1,000 dilutions.

Processing of infected cells for confocal microscopy was performed as we described previously^{44 (link)}. Purification of the LCVs and their labeling prior to permeabilization to localize AnkB on the cytosolic side of the LCV membrane was performed as we described previously^{44 (link)}. For antibody labeling, goat polyclonal anti-L. pneumophila was used at a dilution of 1:500 and detected by Alexa-Fluor 488-conjugated donkey anti-goat IgG (Invitrogen, Carlsbad, CA). Poly-ubiquitinated proteins were detected using mouse anti-polyubiquitin FK1 antibody at a dilution of 1:50 (BIOMOL International/Affiniti, Exeter, United Kingdom), followed by Alexa-Fluor 647-conjugated goat anti-mouse IgM (Invitrogen, Carlsbad, CA). For detection of 3X-Flag tagged proteins during transfection experiments, mouse monoclonal anti-Flag (Sigma) antibodies were used followed by detection with Alexa-Fluor 488-conjugated donkey anti-mouse (Invitrogen, Carlsbad, CA). An Olympus FV1000 laser scanning confocal microscope was used to examine cells as we described previously. On average, 8–15 0.2 µm serial Z sections of each image were captured and stored for further analyses, using Adobe Photoshop CS3.