Alexa fluor 488 labeled secondary antibody

Bacteria were incubated with 20% NHS (Sigma) in PBS for 30 min at 37 °C and washed three times with PBS. Staining was performed sequentially with goat anti-C3 antibody (Calbiochem, 204869, 1:200), recognizing C3b, followed by Alexa fluor 488 labeled Donkey anti-goat antibody diluted (Invitrogen, A11055, 1:200), each incubated for 30 min followed by washing. Bacteria were fixed in 4% paraformaldehyde and visualized by fluorescence microscopy (Leica or Delta Vision) using 100× objective or analyzed quantitatively by flow cytometry using a Gallios™ flow cytometer (Beckman coulter). For C5b-9 staining exponentially growing bacterial cultures were incubated with 20% NHS (Sigma) followed by incubation with anti-C5b-9 antibody (αE11), (Santa cruz biotechnology, sc-58935, 1:50). Alexa fluor 488 labeled secondary antibody (Life Technologies, A11001, 1:100) was used for further detection. Membrane lipids were stained with Nile red (sigma, N3013). After every incubation bacteria were washed twice with 1X HBSS. Samples were fixed and stained with Nile red for 5 min before spreading the samples on glass slides. Samples were visualized using Leica microscope with 100× objective.

HBMEC was grown on collagen-coated glass slide to confluency. Cells were washed thrice with serum-free medium and then pre-incubated for 30 minutes in experimental medium. Cells were then incubated with E. coli containing a red fluorescence protein (RFP)-expressing plasmid (RS218-RFP), at an MOI of 1:100 for a period of 90 minutes at 37°C with 5% CO₂. Cells were washed with PBS to remove the free, unbound bacteria, and then fixed with 4% paraformaldehyde, permeabilized with Triton X-100 solution, and blocked with 5% BSA in PBS. Cells were then incubated with EGFR antibody overnight at 4°C, washed, and incubated with Alexa Fluor 488-labeled secondary antibody (Life Technologies A11034), followed by nucleus staining with DAPI (Vector Laboratories H-1200). The glass slide was mounted and visualized using fluorescence microscopy.

Cells were plated on poly-D-lysine coated slides (Sigma) and were fixed in 4% paraformaldehyde and permeabilized with Triton 1% X-100. After blocking, cells were incubated with primary antibodies, anti–E-cadherin (Abcam, 1:50) and anti-N-cadherin (Abcam, 1:50), subsequently with Rhodamine-linked goat-anti-mouse IgG1 (Santa Cruz, 1:50) and with Alexa Fluor 488–labeled secondary antibody (Life technologies, 1:500) respectively, and finally stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Zeiss LSM 510 confocal microscope system.

The staining procedure for PCMO has been published earlier [6 (link)]. Briefly, cytospins of PCMO were fixed in 1% paraformaldehyde, blocked and incubated with anti-human CD14 antibody (BD Biosciences, Heidelberg, Germany) at room temperature for 2 h and Alexafluor 488–labeled secondary antibody (Life Technologies) for 1 h. Cells were permeabilized with Triton X-100 (0.5%), incubated overnight with the anti-human Ki67 (BD Pharmingen) followed by Alexafluor 555-labeled secondary antibody (Life Technologies). In all experiments, Ki67-positive cells were counted double-blind by two investigators in at least 4 visual fields per slide and related to the total cell count of CD14-positive monocytes in the same field.