4 6 diamino 2 phenylindole

For immunohistochemical staining, normal and 3-day infected eyes from C57BL/6 and BALB/c mice were enucleated (n = 3/group/time), immersed using ice-cold PBS, embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN, USA), and frozen in liquid nitrogen. Immunohistochemical staining was performed with the UltraSensitive SP Immunodetection Kit (Maixin, Inc., Fuzhou, China) following the manufacturer’s protocol. Primary antibodies (rabbit anti-mouse STING) were purchased from PeproTech (Rocky Hill, NJ, USA). Controls were similarly treated, but the primary antibody was replaced with isotype-matched goat IgG. For immunofluorescent staining, cells were seeded on sterile glass cover slips, cultured overnight, and then fixed with 4% paraform (Sigma) in 4°C. Slips were sequentially incubated with rabbit anti-mouse STING Ab (1:200, PeproTech), rabbit anti-mouse NF-κB (1:200, Cell signaling), and Alexa Fluor 488-conjugated goat anti-rabbit IgG Ab (1:1,000, Millipore, Billerica, MA, USA), followed by incubation with 4,6-diamino-2-phenyl indole (1:10,000, Sigma) for nuclear staining. Controls were similarly treated, but the primary Ab was replaced with isotype-matched IgG. For histopathology, sections were HE stained as described by others (29 (link)). All sections were visualized with a Carl Zeiss microscope (Carl Zeiss, Inc., Oberkochen, Germany).

GnRH analog (GnRH-a), Tetradecanoylphorbol acetate (TPA), Okadaic Acid, Polyethylenimine (PEI), 4′6-diamino-2-phenylindole (DAPI) and PLA kit were obtained from Sigma (Rehovot, Israel). GF109203x was obtained from Calbiochem (Darmstadt, Germany). Protein A/G beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dharmafect was obtained from Thermo Scientific (Lafayette, CO, USA). 8-iso PGF2 α was purchased from Cayman Chemical (Ann Arbor, MI, USA). Monoclonal anti-PP2A Ab was obtained from BD Transduction Laboratories (New Jersey, USA). Anti-IGBP1 and monoclonal anti-PI3K Abs were obtained from Abcam (Cambridge, UK). Anti-GFP and anti-HA Abs were obtained from Roche Diagnostics (Mannheim, Germany). Monoclonal anti-AKT, Tubulin, GAPDH, and PKC α were obtained from Santa Cruz Biotechnology (CA, USA). Abs to phosphorylated JNK (pJNK), general JNK1/2 (gJNK), general AKT (gAKT) and histone H1 were from Sigma (Rehovot, Israel). Anti-phospho AKT (pS473AKT) was obtained from Cell Signaling Technology (Boston, MA, USA). Anti-PI3K was purchased from Upstate (Lake Placid, NY New York), or Millipore (Billerica, MA). The anti-phosphorylated PI3K (P85-S608) Abs was prepared by the Ab unit of the Weizmann Institute of Science (Rehovot Israel as previously described [50 (link), 51 (link)]). Secondary Ab conjugates were from Jackson Immunoresearch (West Grove, PA, USA).

To identify the cell types in the skin that were positive for MC1R, double staining for cell types of interest was performed on nine or ten samples randomly selected from those positive for single MC1R staining. The anti-MC1R monoclonal antibody was reacted in the same manner as described above. Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody (Life Technologies, Carlsbad, CA, USA) was used as the secondary antibody. Subsequently, double staining was performed using the following antibodies against cell-specific markers: anti-prolyl-4-hydroxylase β antibodies (1:50, Acris Antibodies, Herford, Germany) for fibroblasts, anti-CD68 antibodies (1:200, Biolegend, San Diego, CA) for monocytes/macrophages, anti-CD66b antibodies (1:250, Biolegend) for neutrophils, and anti-CD31 antibodies (1:50, R&D Systems) for endothelial cells. Antibodies labeled with Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. For nuclear counterstaining, 4′,6′-diamino-2-phenylindole (Sigma-Aldrich) was used. Immunofluorescence-stained tissue sections were analyzed using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).