4. 8 Immunofluorescence Assay
IFA was performed as described previously (Narwal et al. , 2022).
The PFA-fixed cells were washed twice with PBS, and blood-stage
parasites were permeabilized with 0.1% Triton-X100 in PBS for 10 min
and liver stages in chilled methanol for 20 min at 4°C and blocked with
1% BSA-PBS. The cells were incubated for 1 h with the following primary
antibodies: anti-HA antibody (diluted 1:1,000, Novus Biologicals, USA),
anti-BiP (diluted 1:125, MRA‐1246, BEI resources), anti-UIS4 (diluted
1:1,000 (Mueller et al. , 2005b), anti-merozoite surface protein 1
(MSP1) monoclonal antibody (diluted 1:5000 (Holder and Freeman, 1981),
and anti-acyl carrier protein (ACP, diluted 1:800) (Gallagher and
Prigge, 2010). The signals were visualized with secondary antibodies:
Alexa Fluor 594-conjugated anti-rabbit IgG (diluted 1:500; Invitrogen,
USA), Alexa Fluor 488-conjugated anti-mouse IgG (diluted 1:500;
Invitrogen, USA), and Alexa Fluor 488-conjugated anti-rabbit IgG
(diluted 1:500; Invitrogen, USA). The nuclei were stained with Hoechst,
and the coverslips were mounted using Prolong Diamond antifade reagent
(Thermo Scientific, USA). Images were acquired using FV1000 software on
a confocal laser scanning microscope (Olympus BX61WI) equipped with a
UPlanSAPO 100x/1.4 oil immersion objective. The EEFs were counted
manually, and the area was measured using Nikon NIS elements BR imaging
software under an Eclipse 80i fluorescence microscope/Plan Fluor
40x/0.75 objective.