Evaluation of mouse polyclonal antibody against nNav1.5 to recognize nNav1.5 protein in human breast cancer cells using fluorescence immunostaining

Abstract

Abstract Background Neonatal splice variant of Nav1.5 (nNav1.5) is a known tumor-associated antigen (TAA), re-expressed in adult aggressive breast cancer/tumor biopsies positive for lymph node metastasis. Accurate assessment of nNav1.5 protein in breast cancer cells/tissue is important in validating its potential value as a prognostic or predictive marker. Mouse polyclonal antibody (pAb) against nNav1.5 has been obtained. Herein, the ability of the pAb to selectively recognize nNav1.5 protein was evaluated on a panel of cell lines using fluorescence immunostaining. Methods Mouse mammary cell line, 4T1, human breast cancer cell lines, MDA-MB-231, and MCF-7 as well as human non-cancerous epithelial cell line, MCF-10A were acquired (ATCC) and maintained. Accordingly, the cells were cultured in its respective complete media supplemented with 5% fetal bovine serum (FBS), L-glutamine (4mM) and penicillin-streptomycin (1%) at 37 °C in a 5% CO2, relatively humidified atmosphere. For fluorescence immunostaining, the cells were seeded overnight on a cover-slip in 24-well culture plate, fixed, permeabilized and stained with primary pAb followed by incubation with secondary antibody conjugated with AlexaFluor-555. Fluorescence microscopy was conducted using Leica Inverted DMi8 Fluorescence and captured using DFC 365 FX Camera. Results Red fluorescence (secondary AlexaFlour-555) signals were prominent in aggressive human breast cancer cells, MDA-MB-231 and aggressive mouse mammary cancer cells, 4T1; both cell lines are known as positive for nNav1.5 expression. On the contrary, in less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A which lacks nNav1.5 expression, red fluorescence signals were not detected similar to the no-primary and no-primary/secondary controls. Conclusions The obtained mouse pAb against nNav1.5 could serve as a useful tool in validating nNav1.5 protein expression in breast cancer cells. Legal entity responsible for the study The authors. Funding Ministry of Energy, Science, Technology, Environment and Climate Change, Malaysia (Science Fund CIPPM/6113610). Disclosure All authors have declared no conflicts of interest.