Immunofluorescence Assay Protocol for Malaria

Cells were fixed using a mixture of 4% paraformaldehyde and 0.015% glutaraldehyde and permeabilized with 0.1% Triton-X100 as described previously^{62 (link)}. Primary antibodies used for IFAs in this study were the following: mouse anti-MSP1 antibody (European Malaria Reagent Repository, 1:500), rat anti-PfBiP MRA-1247 (BEI Resources, NIAID, NIH, 1:100) and mouse anti‐HA(6E2) (Cell Signaling Technology Inc., 1:100). Anti-mouse and anti-rat antibody conjugated to Alexa Fluor 488 or Alexa Fluor 546 (1:100, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) were used as secondary antibodies. Cells were mounted on ProLong Gold with 4′,6′-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA) and imaged using a Delta-Vision II microscope system with an Olympus IX-71 inverted microscope using a 100 × objective A. Image processing, analysis and display were preformed using DeltaVision softWoRx software version 7.0.0 (GE Healthcare Life Sciences) and Adobe Photoshop 21.2.0 (https://www.adobe.com/products/photoshop.html). Adjustments to brightness and contrast were made for display purposes.