Titermax gold adjuvant liquid

For EhADH immunodetection, we obtained rabbit polyclonal antibodies (α-EhADH) against a specific EhADH peptide (N-566 QCVINLLKEFDNTKNI 582-C) localized within the adherence domain. New Zealand male rabbits were immunized three times (each 2 weeks) with 300 μg of this peptide diluted in TiterMax® Gold Adjuvant liquid (Sigma). Other primary antibodies used were: mouse against EhADH (mAbAdh) (Arroyo and Orozco, 1987 (link)), α-actin (kindly donated by Dr. José Manuel Hernández from Department of Cellular Biology, CINVESTAV, Mexico), α-claudin-1 (Invitrogen), α-occludin (Invitrogen) and α-caveolin-1 (Santa Cruz Biotechnology); rabbit α-ZO-2 (Invitrogen), α-ZO-1 (Invitrogen), α-occludin (Invitrogen), α-α/β tubulin (Cell Signaling), and α-PCNA (Azuara-Liceaga et al., 2018 (link)); and goat α-clathrin (Santa Cruz Biotechnology) and α-GAPDH (Santa Cruz Biotechnology). For some experiments, mouse IgM isotype control (Thermo Fisher) was used. Secondary antibodies included: α-rabbit, α-mouse and α-goat HRP-labeled IgG (1:10,000) (Life technologies); and α-rabbit, α-mouse and α-goat FITC-, TRITC- and Cy5-labeled IgM, and IgG (1:100) (Zymed) antibodies.

For EhADH immunodetection, we obtained hamster polyclonal antibodies (α-EhADH) against a specific EhADH peptide (N₅₆₆-QCVINLLKEFDNTKNI-C₅₈₂) localized within the adherence domain. Male hamsters were immunized three times (each 2 weeks) with 300 μg of this peptide diluted in TiterMax^® Gold Adjuvant liquid (Sigma), then, animals were bled and antibodies obtained. Other primary antibodies used were: rat α-EhVps23 (Galindo et al., 2021 (link)), mouse monoclonal α-HA (GeneTex), mouse α-EhVps32 (Avalos-Padilla et al., 2015 (link)), mouse monoclonal α-Ubiquitin (Santacruz), rabbit α-EhCP112 (García-Rivera et al., 1999 (link)) and mouse monoclonal α-human actin (kindly given by Dr. Manuel Hernández, Cell Biology Department, CINVESTAV) antibodies. Secondary antibodies were: HRP-labelled α-rabbit, α-mouse, α-hamster and α-rat IgGs (Zymed) for western blot. FITC-labelled α-rabbit and α-mouse IgGs, Alexa 647 α-hamster and TRITC-labelled or Alexa 405 α-rat IgGs (Life Technologies) for immunofluorescence. For immunoelectron microscopy experiments, we used α-rat IgG, conjugated with 10 nm gold particles (TED Pella Inc).