Anti icam 1 antibody

Refined storax oil was purchased from Tianjin ZHONGXIN Pharmaceutical Group Limited by Share Ltd Darentang Pharmaceutical Factory and conformed to the standard of China Pharmacopeia (2010 version). Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), glucose-free DMEM, fetal bovine serum (FBS), HBSS, and 0.25% Trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). CytoTox-ONETM Homogeneous Membrane Integrity Assay and Cell Counting Kit-8 (CCK-8) were purchased from Promega (Madison, WI, USA) and Dojindo (Kumamoto, Japan), respectively. DCFH-DA and Trizol were obtained from Invitrogen (Eugene, USA). Penicillin and streptomycin were purchased from Beyotime (Shanghai, China). Anti-NF-κB p65, anti-p-IκBα antibody, anti-p-IKK antibody, and anti-β-actin antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-LaminB1 antibody, anti-iNOS antibody, anti-IL-1β antibody, anti-ICAM-1 antibody, and anti-VCAM-1 antibody were purchased from Abcam Technology (Cambridge, MA, USA). SYBR® Select Master Mix and High Capacity cDNA Reverse Transcription Kits were obtained from Applied Biosystems (Foster City, USA).

The uteri, thoracic aortas and breast tissues from the different groups were collected and fixed in 4% paraformaldehyde overnight, dehydrated using a series gradient of ethanol, carefully embedded in paraffin and sectioned into 5‐μm‐thick slices. After deparaffinization in xylene and hydration with a series gradient of ethanol, sections of the tissues were incubated with 3% H₂O₂ for 10 minutes, followed by three PBS washes. Antigen retrieval from the samples was conducted by microwave treatment in citrate buffer (pH 6.8). Then, sections were separately incubated with primary antibodies: anti‐IGF‐1R receptor antibody (1:200) (Abcam) and anti‐ICAM‐1 antibody (1:200) (Abcam) at a constant temperature of 4°C overnight. After washing three times with PBS, sections were probed with the corresponding secondary antibody using a PV‐9000 polymer detection kit (Zhongshan), and immunoreactivity was visualized using 3,3‐diaminobenzidine (DAB). After counterstaining with haematoxylin, sections were observed under a light microscope (Olympus).

Proteins from tissues were extracted using tissue lysates. Proteins were separated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skim milk and incubated overnight with an anti-ICAM1 antibody (Abcam) at 4°C. The membrane was then incubated with a secondary antibody (1:5000) for 1 hr. The protein band was visualized using a fluorescent kit (P0018S, Beyotime) and a chemiluminescence imaging system (T-4600, Tanon). Protein bands were quantified with ImageJ; samples were normalized to β-actin protein levels.

As we described previously^{18 (link),19 (link)}, the left lung tissues (50 ug) were homogenized in RIPA buffer (catalogue number: 89900; Thermo Scientific, USA) containing phosphatase inhibitor cocktail (catalogue number: 04906845001; Roche Applied Science, USA) and protease inhibitor cocktail (catalogue number: P2714; Sigma, USA). Homogenates were centrifuged at 4 °C (13,000 rpm, 20 min). The supernatant was absorbed as the total protein. The protein concentration was measured by BCA protein assay according to the manufacturer’s instructions (catalogue number: 23227; Pierce Biotechnology, USA). Twenty microgram proteins each lane were loaded on a polyacrylamide gel and transferred onto polyvinylidene difluoride membrane. Then the proteins in membrane were incubated with the primary antibodies at 4 °C: anti-AQP1 antibody (1:1000, catalogue number: ab168387; Abcam, USA), anti-ICAM-1 antibody (1:1000, catalogue number: ab206398; Abcam, USA), anti-ZO-1 antibody (1:1000, catalogue number: ab190085; Abcam, USA), anti-MMP2 antibody(1:1000, catalogue number: ab37150; Abcam, USA), anti-MMP9 antibody(1:1000, catalogue number: ab38898; Abcam, USA), anti-β-Actin antibody (1:5000, catalogue number: ab50591; Abcam, USA). The protein bands were developed by enhanced chemiluminescence (Pierce, USA). The intensities of AQP1, ICAM-1, ZO-1, MMP2, MMP9 proteins were normalized by those of β-Actin.