Alexafluor594 labeled goat anti mouse

Acetaminophen (APAP), collagen, penicillin/streptomycin, Williams’ Medium E, hydrocortisone hemisuccinate, insulin, dimethyl sulfoxide (DMSO), DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride), Mowiol and 6-carboxyfluorescein diacetate were purchased from Sigma-Aldrich (Steinheim, Germany); fetal bovine serum and glutamine were purchased from Biochrom (Berlin, Germany). For immunofluorescence staining, the following antibodies were used: anti-ZO1 mouse IgG (BD Biosciences, Heidelberg, Germany), anti CYP2E1 rabbit and anti CYP3A4 rabbit IgG (Bioss Antibodies, Woburn, MA, USA), AlexaFluor594 labeled goat anti-mouse and AlexaFluor488 labeled goat anti-rabbit antibody (Invitrogen, Darmstadt, Germany). A live/dead cell staining kit II was purchased from Promocell.

Collagen type II (COL2) distribution in the IVD was analyzed by immunofluorescence staining. Antigen retrieval was performed in paraffin sections through incubation with 20 μg/mL proteinase K (Sigma-Aldrich) solution for 15 min at 37 °C. Sections were incubated overnight, at 4 °C, with anti-collagen II-II6B3 (1:20, Developmental Studies Hybridoma Bank at the University of Iowa, Department of Biology, Iowa City, IA, USA) antibody, followed by incubation with Alexa Fluor 594-labeled goat anti-mouse (1:1000, Invitrogen) antibody. Sections were mounted in Fluorshield with DAPI (Sigma-Aldrich). Representative images of the slides were taken using fluorescence microscopy (Axiovert 200 M, Zeiss, Oberkochen, Germany). The percentage of COL2 in NP was determined as described by Cunha et al. [11 (link)].