Image studio lite 4

Sera protein concentration was measured with Pierce^TM BCA Protein Assay (Thermo Fisher Scientific, Les Ulis, France). Protein extracts were separated on 4–12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific), and transferred to a nitrocellulose membrane using the iBlot2 Dry Blotting system (Thermo Fisher Scientific) for 8 min 30 at 20 V. Total protein on blotted membranes were determined with the Revert 700 Total Protein (LI-COR Biosciences, Lincoln, NE) staining. Membranes were washed, blocked in Odyssey Blocking Buffer (LI-COR), 1 h, room temperature. Primary antibody (MYOM3 17692−1-AP, Proteintech 1/500 in 50% blocking buffer) was incubated overnight at 4 °C. After washings in 1× TBST (Tris/HCl (pH 7.5—20 mM)/NaCl (150 mM)/Tween 20 (0.1%)), the diluted secondary antibodies (1/1000 in 50% Odyssey Blocking Buffer) were incubated 1 h at room temperature. Membrane images were acquired with the Odyssey Infrared Imaging System. Band density was quantified using Image Studio Lite 4.0 software (LI-COR Biosciences, Lincoln, NE, USA) and normalized to total protein values.

Mice bearing MKN45P-luc peritoneal tumors were intraperitoneally injected with ZAS-QDs (30 nmol Zn per g body-weight) or ZHS-QDs (45 nmol Zn per g body-weight) in PBS (500 μl) with or without 450 μg of iRGD. At 70 min postinjection of QDs, the mice were intraperitoneally injected with luciferin (15 mg ml⁻¹ in PBS, Biosynth International, Itasca, IL) at a dose of 0.28 mg per g body-weight. The mice were then anesthetized with isoflurane, and imaged at different time points for luminescence with a Xenogen IVIS imager (Perkin-Elmer) and for NIR with a Li-Cor Pearl Impulse imager under 800 nm channel. Some mice also had an extraperitoneal subcutaneous MKN45P-luc tumor in the presence of the peritoneal tumors. At 90 min postinjection of QDs, 1× Ag-TS (400 μl) was intraperitoneally injected. After 5 min, the mice were imaged with the Xenogen IVIS and Li-Cor Pearl Impulse imagers. The mice were then immediately sacrificed under deep anesthesia by cardiac perfusion with PBS, and the necropsied mice (in situ imaging) and resected tissues (ex vivo imaging) were imaged again. The abdominal cavity of the necropsied mice was not washed before in situ imaging. The tissues were processed for immunofluorescence as described elsewhere^{33 (link)}. Fluorescence intensity quantification was performed with Li-Cor Image Studio Lite 4.0 software.

Retinas were homogenized in PBS with protease inhibitors (cOmplete, mini, EDTA-free cocktail tablets, Roche) and centrifuged at 10,000 g for 5 min to remove cellular debris. Protein concentrations were determined using total protein assay (Pierce 660 nm Protein Assay Kit, Thermo Fisher Scientific) and then retinal lysates (150 μg) were incubated with cytokine array membranes (Panel A, R&D System) according to the manufacturer’s protocol. Membranes were developed with 800CW streptavidin (IRDye, LI-COR) for 30 min, and imaged using a LI-COR Odyssey system. Images were analyzed with Image Studio Lite 4.0 software (LI-COR). Fluorescence intensities for each cytokine were averaged and normalized relative to healthy normal retinas.

Total protein lysates were isolated from frozen rat tissue samples using RIPA buffer with Halt Protease- und Phosphatase-Inhibitor-Cocktail (100X) (ThermoFisher Scientific, Waltham, MA, USA) Concentration was measured using Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Proteins (40–60 μg) were separated on a NuPAGE 4–12% w/v Bis-Tris 1.0-mm minigel (Invitrogen, Carlsbad, CA) and detected by primary antibody anti-Cre polyclonal rabbit Antibody (NB100-56135) according to the manufacturer’s instructions (dilution 1:1000). An anti-Gapdh mouse monoclonal antibody was used as a control (dilution 1:5000). IRDye 800CW and IRDye 680 (LICOR Bioscience, Lincoln, NE, USA) were used as secondary antibodies (Dilution 1:10.000). Protein bands were visualized using the Odyssey Infrared Imaging System and quantified using Image Studio Lite 4.0 software (both LI-COR Biosciences, Lincoln, NE, USA).

Total protein lysates were isolated from frozen mouse hippocampal tissue samples using RIPA buffer with cocktail protein inhibitors (ThermoFisher Scientific, Waltham, MA, USA). Proteins (30–60 μg) were separated on a 12% SDS-PAGE gel and detected by primary antibodies (Supplementary Table S2) according to the manufacturer’s instructions. An anti-Gapdh mouse monoclonal/rabbit polyclonal antibody was used as a control. IRDye 800CW and IRDye 680 (LICOR Biosciense, Lincoln, NE, USA) were used as secondary antibodies (Supplementary Table S2). Protein bands were visualized using the Odyssey Infrared Imaging System and quantified using Image Studio Lite 4.0 software (both LI-COR Biosciences, Lincoln, NE, USA).

The relative levels of 40 different cytokines were determined using mouse cytokine antibody array (Panel A, R&D System), according to the manufacturer’s protocol. Retinas were homogenized in PBS with protease inhibitors (cOmplete, mini, EDTA-free cocktail tablets, Roche). Samples were centrifuged at × 10,000g for 5 min to remove cellular debris, and Triton X-100 was added to a final concentration of 1%. Protein concentrations were quantified using total protein assay (Pierce 660 nm Protein Assay Kit, Thermo Fisher Scientific). Pre-mixed sample/antibody cocktails were incubated with blocked membranes overnight at 4 °C on a rocking platform shaker. After 3 washes, membranes were incubated with 800CW streptavidin (IRDye, LI-COR) for 30 min and washed 3 times before being scanned on the LI-COR odyssey imaging system. Data were analyzed with image studio lite 4.0 software (LI-COR). The average fluorescent intensity of each cytokine was quantified for each retinal sample; the light-dependent changes in cytokine expression were determined by dividing the light-exposed values by those of the dark-reared samples processed in parallel from the same genotypes (WT and Arr1^−/−) and performed in triplicate.

To verify whether transfection with pSELECT-GFPzeo-TET1 or pSELECT-GFPzeo-TET2 vectors results in the production of the respective TET proteins, HEK293 cells were suspended in 500 µL of RIPA buffer containing protease inhibitor mix, incubated on ice for 10 min, and then centrifuged at 400× g for 20 min at 4 °C. The protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Twenty micrograms of the extract were used for electrophoresis and transferred to the membrane following standard procedures. The membrane was blocked and incubated with anti-TET1 (1:1000; Abcam, Cambridge, UK), anti-TET2 (1:500, Active-Motif, Carlsbad, CA, USA), or anti-GFP (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) primary antibodies, washed in TBST and incubated with the appropriate secondary antibodies. Signals were detected using the Clarity ECL Western Blotting Substrate (Bio-Rad, Hercules, CA, USA) and analyzed using a GeneGnome chemiluminescence imaging system (Syngene, Cambridge, UK) and Image Studio Lite 4.0 software (LI-COR, Lincoln, NE, USA) (Supplementary Figure S1).