Cyanine3 (cy3)

ICF was performed to visually examine the changes on protein localization and distribution in HaCaT cells after RNAi alone or with drug treatment. HaCaT cells were cultured on glass coverslips placed in 24-well dishes. After treatment cells were washed with PBS twice, fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 8 min. After blocking with 1% bovine serum albumin, cells were incubated with target primary antibody at an appropriate dilution at 4 °C overnight. On the next day, cells were incubated with a secondary antibody conjugated with Cy3 (Invitrogen) for 30 min, and then mounted by using Prolong Gold Anti-fade Reagent with DAPI (Invitrogen) for nuclei staining. Immunofluorescence micrographs were obtained by using an Olympus FSX100 Fluorescence Microscope (Olympus, Tokyo, Japan).

Embryos were fixed in two different ways. For staining with anti-Crumbs (Cq4) and anti-Armadillo (N27A1) antibodies (both from the Developmental Studies Hybridoma Bank), we fixed the dechorionated embryos by a two second heat treatment at 93.4 °C, basically as described in Miller, Field & Alberts (1989) (link). For the anti-Coracle (C566.9) and anti-Fasciclin 3 (7G10) stainings (antibodies also from the Developmental Studies Hybridoma Bank), we fixed dechorionated embryos according to Krahn et al. (2010) (link). The rest of the protocol was according to Miller, Field & Alberts (1989) (link). Anti-Crumbs was used at a 1:20 dilution, anti-Coracle at a1:200, anti-Fasciclin 3 at 1:200, and anti-Armadillo at a1:20. Secondary antibody was anti-mouse conjugated with Cy-3 (Invitrogen) at 1:1,000. For nuclei, we used Sytox-Green (1:3,000) according to the manufacturer’s instructions (Invitrogen). Embryos were mounted in Vectashield (Vector Laboratories), and imaged using a Zeiss 780 confocal microscope with 25× and 63× objectives. Images were acquired with Zeiss software, and manipulated using ImageJ (NIH). We used stage 13–14 embryos for the Crumbs, Coracle, and Armadillo antibody stainings; for the Fasciclin 3 staining, we used stages 15–16 embryos, except for Fig. S1, where stages 13–14 embryos were used. Finally, in Figs. 1C and 2 stage 16 embryos were used.

The cells were cultured on an AFS-coated 8-chamber polystyrene vessel tissue-culture-treated glass slide (CORNIG Inc., Corning, ME, USA). After treatment with 600 μM H₂O₂, in the presence or absence of either FAD012 or FA for 1 h, cells were fixed with formalin for 10 min, treated with 0.1% Triton for 10 min, and then blocked with 5% normal goat serum (Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 h. After washing with 0.01 M PBS, the cells were reacted with a primary antibody (eNOS; 1:100, sc-376751, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The next day, after washing with PBS, the cells were reacted with a secondary antibody (Cy3, 1:250, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. After washing with PBS, the nuclei were stained with 1 μg/mL of DAPI reagent for 5 min and then sealed with a cover glass and observed under a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

For immunohistochemical characterization, mice were first deeply anaesthetized with thiopental sodium (150 mg/kg, i.p.) and transcardially perfused with a fixative (4% paraformaldehyde + 15% picric acid in 0.1 M phosphate-buffer (PB), pH 7.2–7.4). Following brain extraction, coronal sections were cut (50 μm) on a Leica VT1000S vibratome (Leica Microsystems, Vienna, Austria) and immunostained against mGluR5 and D1, based on previously described procedures [76 (link)]. A rabbit antibody against mGluR5 (Frontier Institute, Hokkaido, Japan; AB_2571802) and a goat antibody against D1 (Frontier Institute, AB_2571594) were diluted 1:1000 in 2% normal goat serum (NGS), 0.1% Triton X-100 in Tris-buffered saline (TBS; pH 7.4) and sections incubated for 48 h at 6 °C. Sections were then incubated overnight with the respective secondary antibodies (anti-goat Alexa Fluor™488, 1:1000, Jackson ImmunoResearch Europe Ltd.; anti-rabbit Cy3, 1:500, Invitrogen, ThermoFisher Scientific, Vienna, Austria). After three washing steps with TBS, sections were finally mounted onto gelatin-coated slides and coverslipped with Vectashield (Vector Laboratories, Burlingame, US). Immunofluorescent sections were examined using a Zeiss AxioImager M1 microscope or a confocal laser-scanning microscope (SP5, Zeiss, Oberkochen, Germany) for low and high-resolution image acquisition, respectively.

Monoclonal rat anti–α-tubulin (1:500; AbD Serotec) and rabbit anti-Cdc14 antibody (SC-33628; Santa Cruz Biotechnology, Inc.), Secondary antibodies, pre-absorbed against sera from other species used in labeling were conjugated with Cy3 (Millipore) or Alexa Fluor 488 (Invitrogen) and diluted 1:500 (Cy3 and Alexa Fluor 488). DNA was visualized by staining with DAPI.