Zebrafish Xenotransplant Cancer Model for Drug Screening - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 215

daughter cells, after several doublings, fluorescently labeled single cells were clearly
visible on the cancer cell masses (Haldi et al., 2006).

17.3.4 Cell Transplantation

The transplantation protocol used was similar to the protocol described for homograft
transplantation in zebrafish (Ho and Kane, 1990). CM-DiI-labeled cells were loaded
into a pulled glass micropipette (VWR) that was drawn on an electrode puller and
trimmed to form a needle with

m outer diameter.
The microneedle was attached to an air-driven Cell Tram (Eppendorf) and the tip of
the needle was then inserted into the yolk of 2 or 3dpf zebrafish. Using positive
pressure, pulse time was controlled to deliver

15 m m inner diameter and 18 m

1500 cells in 15 nL volume. Number
of injected cells was standardized by fixing cell density and injection volume. After a
1 h recovery period at 28 C, injected zebrafish were examined under a fluorescence
microscope for the presence of xenotransplant cells located specifically in the yolk sac
and then transferred to 35 C.

17.3.5 Antibody Sources

Sources for antibodies were as follows: HLA, mouse anti-human, and HLA-DR,
mouse anti-human monoclonal antibodies (Invitrogen, Carlsbad, CA), survivin,
rabbit anti-human monoclonal antibody, XIAP, rabbit anti-human monoclonal anti-
body, and Bax, rabbit anti-human polyclonal antibody (Cell Signaling Technology,
Danvers, MA), and Keap1, rabbit anti-human polyclonal antibody (Santa Cruz
Technology, Santa Cruz, CA).

17.3.6 Whole Mount Xenotransplant Zebrafish
Immunostaining

Whole mount xenotransplant zebrafish immunostaining was performed to confirm
antibody specificity to xenotransplant human colon cancer cells and to assess
zebrafish cross-reactivity. Xt zebrafish were processed using Dent's fixative and
whole mount immunochemical staining was performed using human cell-specific
antibodies, followed by staining with Alexa Fluor 488-conjugated secondary anti-
body (Invitrogen). Zebrafish were deposited in methylcellulose and laterally oriented
for image acquisition using a Zeiss Stemi SV11 Apo upright fluorescence microscope
equipped with a Zeiss AxioCam (Zeiss, Thornwood, NY). CM-DiI-labeled cells were
visualized in the rhodamine channel and human antibody-stained zebrafish were
visualized in the FITC channel.

17.3.7 Quantitative Whole Xt Zebrafish ELISA

Xt zebrafish were fixed in Dent's fixative (DMSO/methanol ¼ 1/4) and processed

using a whole mount immunochemical staining protocol (Westerfield, 1993);