Hrp conjugated goat anti rabbit igg secondary antibody

For assessment of protein levels, cells were lysed using RIPA buffer, and 10 µg of total protein was separated and electroblotted as described previously^{28 (link)}. Protein bands were detected using a goat anti-rabbit or mouse IgG-HRP conjugated secondary antibody (200 µg/ml; Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence (Amersham Biosciences). Co-immunoprecipitation was performed as previously described^{39 (link)}. In brief, rabbit anti-CASZ1 antibody was first incubated overnight with Dynabead M-280 Sheep Anti-Rabbit IgG magnetic beads (ThermoFisher Cat. # 11204D). Whole cell extracts from CTRtetCASZ1b cells treated with Dox for 24 h were incubated for 4 h with the CASZ1 antibody-bound magnetic beads under constant shaking at 4 °C. The co-IP products were eluted by incubating with 1x SDS loading buffer heated to 100 ^oC for 3 min. Protein bands were detected using a goat anti-rabbit IgG-HRP conjugated secondary antibody (200 µg/ml; Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence (Amersham Biosciences).

Protein was extracted from cultured cells in a NP-40 buffer (50 nM Tris HCL pH 7.6, 150 mM NaCl, 1% NP-40, 5 mM NaF, 1 m MEDTA; Thermo Fisher) containing a protease inhibitor (Roche) and phosphatase inhibitors (Sigma-Aldrich, St. Louis). Protein samples were run on 10% Bis-Tris gels (NuPage, Thermo Fisher) and transferred to PVDF membranes. The membranes were blocked in 5% nonfat dry milk for 1 hour and then incubated with primary antibody for 3 hours: SRGAP2 (1:1000, Abcam, Cambridge, #ab121977) and β-actin (1:2000, Abcam, #ab8227-50). Subsequently, membranes were incubated in goat anti-rabbit IgG-HRP conjugated secondary antibody (1:5000, Santa Cruz, Dallas, #sc-2004) for one hour. Blots were thoroughly washed and developed using the WesternBright Quantum detection kit (Advansta, Melano Park) and Licor Odyssey Image Studio.

A standard western blot protocol was carried out to evaluate ALK protein expression in cell lysates of NB cell lines. After 48 and 72 hours from transfection, cells were washed twice with ice-cold PBS and lysed with 50-100 μl of lysis buffer (Invitrogen), supplemented with a cocktail of protease inhibitors and PMSF (phenylmethylsulfonyl fluoride) (Sigma-Aldrich). Lysates were vortexed every 10 minutes and incubated on ice, for a total of 30 minutes, and then cleared by spinning. Proteins were separated in Mini-PROTEAN® TGX™ Precast 4-20% Gels (Biorad), and transfer was performed by Trans-Blot Turbo transfer system (Biorad). Membranes were incubated with primary antibodies for ALK (Cell Signaling) and GAPDH (Cell Signaling), which was used as a control for equal protein loading. Goat anti-rabbit IgG HRP conjugated secondary antibody was from Santa Cruz Biotechnologies. Blots were developed with Amersham’s ECL Prime (GE Healthcare) and images acquired by a chemiluminescent detection system (Uvitec Cambridge). Quantification of western blot bands was performed by ImageJ [74 ].

The proteins of sorted GlycoA⁺ NRBC were extracted by lysis buffer (Biotech, Beijing, China). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Samples were separated by 12% SDS-PAGE gels and then transferred to PVDF membranes (PE, USA) by a Trans-Blot Cell system (Bio-Rad, USA) using standard Western blotting procedures. The membranes were probed with a rabbit anti-human TOM20 antibody (Cell Signaling Technology, number 13929, USA) at 1 : 1000 and incubated overnight at 4°C. The rabbit anti-human β-actin antibody (Santa Cruz Biotechnology Inc., sc-47778, USA) at 1 : 5000 was used as loading control. The goat anti-rabbit IgG HRP-conjugated secondary antibody (Santa Cruz Biotechnology Inc., sc-2054, USA) was incubated for 1 hour at room temperature. After washing, an electrochemiluminescence (ECL) reagent (Thermo Scientific, number 32106, USA) was used for chemiluminescence.

All chemicals and solvents used were of analytical grade. Tannic acid (purity 95%) was purchased from ACROS organics (Geel, Belgium, Europe). Bradford reagent was acquired from Bio-Rad (Hercules, CA, USA). Acridine orange, catalase, cerium nitrate hexahydrate (purity 99.999%), dimethyl sulfoxide (DMSO), ethyl diamine, HBSS (Hank’s balanced salt solution), hydrogen peroxide (99.9%), ortho-phthalaldehyde, protease inhibitor cocktail, reduced glutathione (GSH), and the chemical cross-linkers of 1, 1′-carbonyldiimidazole (CDI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DPPP (diphenyl-1-pyrenylphosphine) and H₂DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate) were purchased from Molecular Probes (Eugene, OR, USA). DMEM (Dulbecco’s modified Eagle’s medium) and fetal bovine serum were purchased from Gibco (Carlsbad, CA, USA). Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) was obtained from Inlab (São Paulo, Brazil). HRP-conjugated goat anti-rabbit IgG secondary antibody, mouse anti-COX-2, mouse anti-MMP-1, mouse anti-β-actin and ECL reagent were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA).

Immunohistochemistry (IHC) was performed as previously described [36 (link)]. The ovarian tissues of mice were fixed in 4% formaldehyde and paraffin-embedded using standard procedures. First, consecutive 4-µm sections were cut, deparaffinized with xylenes, rehydrated, and retrieved the antigen in sodium citrate solution (pH 6.0) for 20 min. Then the slides were treated with 3% hydrogen peroxide to quench the endogenous peroxidase and blocked with 1% bovine serum albumin for 30 min to block the nonspecific binding. Next, tissue sections were incubated with primary antibody anti-FTO (Abcam, Cambridge, ab126605, USA; 1:150) overnight at 4 ℃. Finally, wash the slides with PBS three times and incubate the slides with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1000, Santa, Cruze) for another 30 min. Finally, a 3, 3-diaminobenzidine tetrahydrochloride (DAB) (Beyotime, Wuhan, China) substrate kit was applied to detect peroxidase reactivity. According to the manufacturer instructions, we prepared DAB peroxidase substrate in 5 ml ddH₂O in a glass vial. Then, drop the DAB substrate on top of the slides and watch the brown staining. Dip slides into ice plus tap water to stop the reaction and rinse under cold tap water for 5 min.