Goat α rabbit igg

For extraction of cell lysate for analysis, purified blood stages, gametocytes and ookinetes were suspended in whole cell lysis buffer (1XPBS, 1% v/v Triton X-100). Proteins resolved by SDS-PAGE were transferred to a polyvinylidene difluoride membrane. Detection was performed using rabbit α-HA (Cell Signaling Technology) (1:1,000), goat α-GFP (Rockland Chemicals) (1:1,000), and 13.1 mouse monoclonal α-P28 (1:1,000) antibodies. Secondary horseradish peroxidase-conjugated goat α-rabbit IgG, goat α-mouse IgG antibodies (Promega), and donkey α-goat IgG (Abcam) were used at 1: 2,500, 1: 2,500 and 1: 5,000 dilutions, respectively. All primary and secondary antibodies were diluted in 5% w/v milk-PBS-Tween (0.05% v/v) blocking buffer.

Parasite proteins were extracted with 1% Triton in PBS supplemented with protease inhibitors. Protein extracts were boiled under reducing conditions in Laemmli buffer, separated using 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane and detected using the rabbit α-PbP47 and a goat α-GFP (Rockland chemicals) antibodies at 1:100 and 1:1000 dilutions, respectively. Secondary horseradish peroxidase conjugated donkey α-goat IgG (Abcam) and goat α-rabbit IgG (Promega) antibodies were used at 1:5000 and 1:10000 dilutions, respectively. Gametocytes and ookinetes were fixed in 4% para-formaldehyde (PFA) in PBS for 20–25 min, smeared on glass slides and air-dried prior to blocking and antibody staining. Dissected midgut tissues were fixed after blood bolus removal in 4% PFA in PBS for 30 min, washed three times in PBS for 10 min, and blocked and permeabilized in 1% BSA and 0.2% Triton in PBS prior to antibody staining. Rabbit α-PbP47, rabbit α-TEP1^{29 (link)} and mouse α-P28 were used at 1:100, 1:300 and 1:1000 dilutions, respectively. Secondary Alexa Fluor 647 goat α-rabbit and 568 goat α-mouse IgG antibodies (Life technologies) were used at 1:1000 dilution. Images were acquired using a Leica SP5 MP confocal laser-scanning microscope, processed by deconvolution using the Huygens software and visualized with ImageJ.