4.4.4. Immunofluorescence

The cells were seeded into the wells of chambered slides, washed with PBS, fixed with ice cold methanol (10 min), and blocked with 5% normal goat serum in PBS (1 h at RT). A primary antibody recognizing the N-terminus of the human TRPA1 protein (NB110-40763, Novus Biologicals) was diluted in 1% normal goat serum in PBS (1:200) (G9023, Sigma) and applied 1h under agitation at RT. Cells without primary antibody were used as immunospecificity control. After washing with PBS (3 × 5 min), a secondary antibody that was diluted in 1% normal goat serum (1:200) (Cy2 anti-rabbit IgG, 111-225-144, Jackson Immuno Research) was applied for 1 h at RT under agitation. Next, cells were washed with PBS (3 × 5 min), coverslipped with 1–2 drops of antifade mounting medium with DAPI (VECTASHIELD; H-1200), and imaged with a fluorescence microscope (Olympus IX51).
