Mouse anti ha 6e2

Parasites were fixed for IFA using 4% Paraformaldehyde and 0.03% glutaraldehyde, then permeabilized with 0.1% Triton-X100. Primary antibodies used were rat-anti-HA 3F10 (Roche, 1:100), mouse-anti-HA 6E2 (Cell Signaling Technology, 1:100), mouse-anti-V5 TCM5 (eBioscence, 1:100), rabbit-anti-V5 D3H8Q (Cell Signaling Technology, 1:100), and mouse-anti-PfPMV (from D. Goldberg 1:1). Secondary antibodies used were Alexa Fluor 488 and Alexa Fluor 546 (Life Technologies, 1:100). Cells were mounted to slides using ProLong Diamond with DAPI (Invitrogen). Fixed and stained cells were imaged using a DeltaVision II microscope system with an Olympus IX-71 inverted microscope. Images were collected as a Z-stack and deconvolved using SoftWorx, then displayed as a maximum intensity projection. Images were processed using Adobe Photoshop, with adjustments made to brightness and contrast for display purposes.
For imaging of parasite cultures using light microscopy, aliquots of culture were smeared onto glass slides and field-stained using Hema3 Fixative and Solutions (Fisher Healthcare), which is comparable to Wright-Giemsa staining. Slides were imaged using a Nikon Eclipse E400 microscope with a Nikon DS-L1-5M imaging camera. To measure parasite size, images were taken and parasites measured using ImageJ (NIH).

Western blots were performed as previously described [55 (link)]. Briefly, ice-cold 0.04% saponin in 1x PBS was used to isolate parasites from host cells. Parasite pellets were subsequently solubilized in protein loading dye to which Beta-mercaptoethanol had been added (LI-COR Biosciences) and used for SDS-PAGE. Primary antibodies used in this study were rat-anti-HA 3F10 (Roche, 1:3000), mouse-anti-HA 6E2 (Cell Signaling Technology, 1:1000), rabbit-anti-HA 715500 (Thermofisher, 1:100), mouse-anti-V5 TCM5 (eBioscence, 1:1000), rabbit-anti-V5 D3H8Q (Cell Signaling Technology, 1:1000), rabbit anti-PfBiP MRA-1246 (BEI resources, 1:500), rabbit-anti-PfEF1α (from D. Goldberg, 1:2000), and mouse-anti-PfPMV (from D. Goldberg 1:400). Secondary antibodies used were IRDye 680CW goat-anti-rabbit IgG and IRDye 800CW goat-anti-mouse IgG (Li-COR Biosciences, 1:20,000). Membranes were imaged using the Odyssey Clx Li-COR infrared imaging system (Li-COR Biosciences). Images of membranes were processed using ImageStudio, the Odyssey Clx Li-COR infrared imaging system software (Li-COR Biosciences). Densitometry analysis of western blot signal was also performed using ImageStudio (Li-COR Biosciences).

All procedures (TEM and Immunofluoresence) were performed as previously described [14 (link), 15 (link)]. The primary antibodies used in this study were: mouse anti-elav (9F8A9, 1:200, Developmental Studies Hybridoma Bank); mouse anti-Crumbs (Cq4, 1:100, Developmental Studies Hybridoma Bank), rabbit anti-aPKC ζ (zeta) (1:200, SAB4502380 Sigma), mouse anti-HA (6E2, 1:500, Cell Signaling), mouse anti-myc (9B11, 1:500 Cell Signaling), rat anti-Crumbs (1:500) [73 (link)], mouse anti-armadillo (N2 7A1, 1:100, Developmental Studies Hybridoma Bank). The C-terminal peptide KVNKLISRFEGGRPRLC (produced in the laboratory of Dr. Charles Zuker) was used as the antigen in rabbits, and the resulting antibody was used at a concentration of 1:200. Fluorescent conjugated secondary antibodies were obtained from either Jackson ImmunoResearch Laboratories or Life Technologies. Rhodamine or Alexa Fluor 647 conjugated phalloidin (1:200, Life Technologies) were utilized for the detection of F-Actin. Confocal images were captured on a Leica TCS SP5. TEM imaging was conducted with a JOEL 1010 and JOEL 1400. All images were processed in Adobe Photoshop.

Primary antibodies were mouse anti-Myc (9B11) (Cell Signaling Technology), rabbit anti-FLAG (F7425) (Merck), mouse anti-HA (6E2) (Cell Signaling Technology), rabbit anti-human GPVI cytoplasmic tail [46 (link)], mouse anti-human GPVI (11A7) [47 (link)], mouse anti-human GPVI (336A9) (Bernhard Nieswandt, unpublished), mouse anti-human ADAM10 (11G2) (a gift from Eric Rubinstein, Paris, France) [47 (link)] and control mouse IgG1 (MOPC-21) (MP Biomedicals).