Cold water fish skin gelatin

Giardia parasites were iced for 30 min and pelleted at 700 × g for 7 min. The pellet was fixed in PME buffer (100 mM piperazine-N,N′-bis [ethanesulfonic acid], pH 7.0; 5 mM EGTA; 10 mM MgSO₄ supplemented with 1% paraformaldehyde [PFA]; Electron Microscopy Sciences, Hatfield, PA), 100 µM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma-Aldrich), 100 µM ethylene glycol bis (succinimidyl succinate; Pierce), and 0.025% Triton X-100 for 30 min at 37 °C. Fixed cells were attached to polylysine-coated coverslips. Cells were washed once in PME and permeabilized with PME plus 0.1% Triton X-100 for 10 min. After two quick washes with PME, blocking was performed in PME supplemented with 1% bovine serum albumin, 0.1% NaN₃, 100 mM lysine, 0.5% cold-water fish skin gelatin (Sigma-Aldrich). Next, 1:200 diluted Alexa 647–conjugated anti-CWP1 antibody (Waterborne) was added to incubate for 1 h. Cells were washed three times in PME plus 0.05% Triton X-100. Coverslips were mounted with ProLong Gold antifade plus DAPI (Molecular Probes). Images were acquired on a DeltaVision Elite microscope using a 60X, 1.42–numerical aperture objective with a PCO Edge sCMOS (scientific Complementary Metal–Oxide–Semiconductor) camera, and images were deconvolved using SoftWorx (API, Issaquah, WA).

Cells on coverslips were fixed in 4% paraformaldehyde (Sigma) for 1 hr at 4°C, permeabilized with 0.1% Triton-X (Sigma), and blocked with 0.2% cold-water fish skin gelatin (Sigma)/10% FCS (GIBCO). All antibodies were incubated in blocking solution plus 0.01% saponin (Sigma), primary antibodies (TBX3, 1:200 [Abnova]; α-MHC, 1:200; [Abcam]; HCN4, 1:100 [Alomone Labs]; Connexin 30.2, 1:200 [Abcam]; and Connexin 45, 1:200 [Abcam]) for 1 hr at RT, secondary antibodies (anti-sheep Alexa Fluor 633, 1:500 [Life Technologies]; anti-mouse Alexa Fluor 647, 1:500 [Abcam]; and anti-rabbit Alexa Fluor 647, 1:500 [Abcam]) for 45 min at RT with additional phalloidin (1:500; Enzo Life Science) and bisBenzimide (DAPI, 1:2,000; Sigma). The coverslips were mounted with Dako mounting medium (DAKO) to glass slides and analyzed by fluorescence microscopy using a Leica SP-5 confocal laser scanning microscope (Leica Microsystems) or an ELYRA PS.1 LSM 780 microscope (Carl Zeiss).

HEK293T cells were transfected with GRP78‐Myc, Mhp271‐Flag, or both and fixed for 30 min in 4% paraformaldehyde (PFA) in PBS. The fixed cells were permeabilized with 0.1% Triton X‐100 for 10 min and blocked for 1 h in 0.2% cold‐water fish skin gelatin (Sigma). Cells were then incubated with mouse anti‐Myc mAb or rabbit anti‐Flag polyclonal antibodies for 1 h at RT, washed three times with PBS, and incubated for 1 h with secondary antibodies coupled to Alexa Fluor 633 or Alexa Fluor 488 (Invitrogen). The cell nuclei were labeled with DAPI (1:1000, Sigma). Fluorescence images were acquired using a confocal microscope (Zeiss, LSM800).