Sp2 aobs confocal laser scanning microscope

Confocal microscopy of melanoma cells transfected with pcDNA3.1-gL and either pME18s-gH[WT] or gH mutant constructs was performed as previously described [27] (link). For infection, melanoma cells were inoculated with 500 plaque forming units (PFU) of recombinant virus. For TK-GFP-BAC transfection, melanoma cells were transfected with pOka-TK-GFP-gB[Y881F], -gH[Δ834-841], and -gB[Y881F]/gH[Δ834-841] BACs using Lipofectamine 2000. Cells were fixed with 4% paraformaldehyde at post 24 hours for non-BAC transfected and infected cells and 72 hours for BAC transfected cells. Cells were probed for gH, early endosomes and the trans-Golgi-network using anti-gH mAb SG3 (mouse), anti-EEA1 mAb (rabbit; Novus Biological), and anti-TGN46 pAb (sheep; AbD Serotec), respectively. TK-GFP-BAC-transfected cells were probed with anti-IE62 (mouse; Chemicon International) and anti-ORF23 (polyclonal rabbit; [36] (link)). Primary antibodies were detected with secondary antibodies, anti-mouse Alexa Fluor 555 (Invitrogen), anti-rabbit Alexa Fluor 488 (Invitrogen), and anti-sheep Alexa Fluor 647 (Invitrogen). Nuclei were stained with Hoechst 33342 (Invitrogen). Images were captured with a Leica SP2 AOBS Confocal Laser Scanning Microscope. Channel merging and image processing was performed with ImageJ and Photoshop.

After 3 days of ABA treatments, DR5::GFP seedlings were stained briefly (50 s) with 10 μM propidium iodide. GFP and propidium iodide fluorescence was then detected using a Leica SP2-AOBS confocal laser scanning microscope and the images were electronically superimposed using LCS Lite software (Leica, Germany). Quantification of the GFP fluorescence signal was performed using ImageJ (National Institutes of Health, United States).

Sections of 3 μm were prepared from paraffin-embedded samples of E. tenella-infected chicken caeca (144 h p.i.) and T. gondii-infected cat intestine (day 7). Deparaffinisation, antigen retrieval and immunofluorescence analysis were carried out as described previously [17 (link)], using antibodies described in the Results section. Alternatively, unsporulated oocysts were vortexed with an equal volume of glass beads (710–1,180 μm, Sigma) and air-dried onto glass slides prior to immunofluorescence assays. Alexa Fluor® 488 goat anti-mouse (green) and Alexa Fluor® 594 goat anti-rabbit (red) secondary antibodies were used at a dilution of 1 in 500 for detecting mouse and rabbit primary antibodies, respectively, and DAPI counterstaining was used at 1 μg/ml. Samples were mounted in VECTASHIELD® Mounting Medium (Vector Laboratories). Epifluorescence imaging was performed using a Leica DMI 6000 B microscope and the Leica LAS AF software. Confocal imaging was performed using a Leica SP2 AOBS confocal laser-scanning microscope and the Leica Confocal software for data collection, with subsequent deconvolution using the Huygens Essential (Scientific Volume Imaging B.V.) software.