Delta vision 2 microscope system

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.

For IFAs, cells were fixed as described previously (60 (link), 61 (link)). The antibodies used for IFA were as follows: rat anti-HA antibody (clone 3F10; Roche) (1:100), mouse anti-AMA1 (1F9 from Alan Cowman), rat anti-PfGRP78 (MRA-1247; BEI Resources, NIAID, NIH) (1:100), mouse anti-MSP1 (12.4; European Malaria Reagent Repository) (1:500), rat anti-AMA1 (28G2; Alan Thomas via BEI Resources, NIAID, NIH) (1:500), mouse anti-EBA175 (R218; B. Kim Lee Sim via BEI Resources, NIAID, NIH) (1:500), and mouse anti-RAP1 (2.29; European Malaria Reagent Repository) (1:500). Secondary antibodies used were anti-rat antibody conjugated to Alexa Fluor 488 or Alexa Fluor 546 and anti-rabbit antibody conjugated to Alexa Fluor 488 (Life Technologies) (1:100). Cells were mounted on ProLong diamond with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and imaged using a Delta-Vision II microscope system with an Olympus IX-71 inverted microscope using a 100× objective or an Elyra S1 SR-SIM microscope (Zeiss). Image processing, analysis, and display were performed using SoftWorx or Axiovision and Adobe Photoshop. Adjustments to brightness and contrast were made for display purposes.