Bovine serum albumin (bsa)

The effects of BEOs on the expression of important proteins involved in the inflammatory response in RAW 264.7 macrophages were assessed by Western blotting. The cells (4 × 10⁵ cells/well in 6-wells plate) were cultured for 24 h before incubating with BEOs for 2 h. The cells were then stimulated by LPS (1 µg/mL) for 24 h before being harvested and resuspended in radioimmunoprecipitation assay lysis buffer (Thermo Scientific, Rockford, IL, USA) to obtain the total protein. Total protein samples (20 µg each) were analyzed using SDS-PAGE before being transferred to a PVDF membrane. The membrane was blocked with T-TBS (Tris-buffered saline, 0.1% Tween 20) containing 2% bovine serum albumin (Biobasic, Markham, Ontario, Canada) for 1 h. The membrane was then incubated with primary antibodies (1:1000 dilutions) for detecting β-actin, iNOS, COX-2, IκBα, p-IκBα, NF-κB p65, and p-NF-κBp65 for 16 h at 4 °C. The membranes were then washed thrice with T-TBS before being incubated with HRP-conjugated goat anti-rabbit IgG for 1 h at 25 °C. The membranes were washed thrice and then incubated with Clarity Max^TM Western ECL substrate (Bio-Rad, Milan, Italy) and the signals were detected using a ChemmiDoc^TM imaging system (Bio-Rad, Hercules, CA, USA). Protein quantities from Western blot results were calculated by Image Lab software Version 6.1 (Bio-Rad, Hercules, CA, USA).

The procedures were modified from previous studies by our group [25 (link), 34 (link)]. After fixation, tissues were permeated with methanol at -20°C overnight and then infiltrated with Tissue-Tek O.C.T. compound (Sakura, Torrance, CA, USA) overnight at 4°C. After cryosectioning, sections (5-μm) were kept at -20°C.
For double staining, cryosections were rinsed by PBS and then pre-incubated with PBS containing 5% bovine serum albumin (Bio Basic, Markham, Canada) for 30 min. Afterward, cryosections were incubated with FXYD12 antibody (400× dilution in PBS) at 4°C overnight. Next, sections were incubated with the secondary antibody (Alexa-flour-546 antibody) for 2 h. Subsequently, sections were incubated with NKA antibody (200× dilution in PBS) for 2 h followed by the secondary antibody (Alexa-flour-488 antibody) for 2 h. The immunoreactions were observed using a laser scanning confocal microscope (FV1000; Olympus) or a fluorescent microscope (BX50; Olympus) with DP72 camera (Olympus) as described in the previous paragraph.

Cytoplasmic and nuclear fractions of cultured cells and mouse tissues were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, IL, USA). The proteins in each fraction were blotted and their relative expression levels were determined as previously described [34 (link),35 (link)]. Briefly, the primary antibodies used in this study were immunoglobulins against COX-2 (Cell Signaling Technology, Danvers, MA, USA), NF-κB (Bioworld Technology, St. Luis. MN, USA), iNOS (Enzo Life Sciences, Farmingdale, NY, USA), HO-1 (Abcam, Cambridge, UK), Nrf2 (Abcam, Cambridge, UK), β-actin, and Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA). They were used at a dilution ratio of 1:1000 in 1% bovine serum albumin (Bio Basic Inc., Markham, ON, Canada) in tris-buffered saline (TBS). After allowing the appropriate secondary antibody (horse radish peroxidase–conjugated) at 1:2000 in TBS to interact with the primary antibody, protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Cheshire, United Kingdom) and LAS4000 Mini (GE Healhcare Life Sciences, Little Chalfont, UK). The digitalized blot images were then densitometrically analyzed using Image-Studio Lite version 5.2 (LI-COR Biotechnology, Lincoln, NE, USA).

Flies were housed at 25°C under an LD cycle on standard media, unless otherwise noted. At each time point ∼10 intestines from female flies <14 days were dissected in PBS (Fisher) and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and then counterstained with DAPI (Thermo Fisher Scientific, 1:5,000) in PBS-T (PBS + 0.2% Triton X-100, Fisher). Intestines were then blocked in 1% BSA (Bio Basic) + 0.2%Triton X-100 (Fisher) and incubated in the same at room temperature for 2 hr with primary antibodies: mouse anti-Delta (Developmental Studies Hybridoma Bank [DSHB], 1:50), mouse anti-prospero (DSHB, 1:50), mouse anti-histone (Millipore, 1:2000), or rabbit anti-PER (generously provided by Patrick Emery, 1:1,500), then incubated at room temperature for 1 hr in secondary goat anti-mouse/rabbit antibodies (Life Technologies, 1:2000), and counterstained with DAPI (Thermo Fisher Scientific, 1:5,000). Samples were imaged using a slide scanner (Zeiss Axio Scan.Z1) that assembled single images consisting of merged and tiled z stacks of the entire tissue sample in a single plane of focus, or by confocal microscopy (Olympus IX81 FV1000) with a 60× water-immersion lens. Images were analyzed using Zen Blue Edition software (Zeiss) and processed using Photoshop CS5 (Adobe).