Mouse monoclonal igg

Cells were plated on poly-L-Lysine-coated round glass coverslips and allowed to attach for 24 h in 24-well plates. Each group was incubated in hypoxic conditions with or without HP. Primary antibodies (CD11b: mouse monoclonal IgG, Abcam, UK; GFAP: mouse monoclonal IgG, Santa Cruz, USA; ferritin: rabbit polyclonal IgG, Santa Cruz, USA) were used at 1:200, and the appropriate secondary antibody (goat poly-IgG to rabbit and donkey poly-IgG to mouse, Abcam, UK) conjugated with Alexa Fluor 488, which was used at 1:400 dilution. Cells were stained as described previously [19]. Briefly, after washing with HBSS, the cells were incubated with the primary antibody for 30 min, followed by washing and incubation with the appropriate secondary antibody for 30 min. For surface antigens such as CD11b, the cells were then fixed with 95% ethanol and 5% acetic acid for 15 min at 4 °C. Intracellular antigens (GFAP, ferritin) required cell permeabilization before incubation with the primary antibody [24]. After washing in PBS, the coverslips were mounted in antifade mounting medium containing DAPI (Life Technologies, USA) and analyzed using a fluorescence microscope.

All 137 RB patients could divided into three groups according to the genotype at rs937283. We randomly selected 8 RB tumor tissues from each groups and then isolated protein. Frozen tumor tissues from RB patients were homogenized on ice and lysed in RIPA buffer containing protease inhibitor (cOmplete, Sigma, CA, USA) and PMSF. The homogenate were sonicated and centrifuged at 12,000 rpm at 4 °C for 5 min to remove cell debris. A BCA assay kit (Beoytime Biotech, Shanghai, China) was used to determine protein concentration and extracted proteins were separated by 8% SDS-PAGE, then separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membrane was blocked with 5% skim milk in TBST (TBS buffer with 0.1% Tween-20), and then incubated with human MDM2 antibody (1:1000 dilution, sc-5304, Mouse Monoclonal IgG, Santa Cruz, USA), p53 antibody (1:1000 dilution, ab28, Mouse Monoclonal IgG, Abcam USA) and GAPDH antibody (1:1000 dilution, sc-32233; Mouse Monoclonal IgG, Santa Cruz, USA) at room temperature for 2 h. Subsequently, horseradish peroxidase(HRP) -conjugated anti-mouse IgG were used as secondary antibody (1:5000 Dilution, Beoytime Biotech, Shanghai, China). Signals were captured by a CCD camera image system (Bio-Rad, CA, USA) with an HRP Chemiluminescent kit (Beoytime Biotech, Shanghai, China).

Extracted rabbit ChP were fixed in 2.5% glutaraldehyde (Merck) in 0.15 M sodium cacodylate (pH 7.4, Sigma-Aldrich, Steinheim, Germany), embedded in Epon (Agar Scientific Ltd, Stansted, Essex England) and ultrasectioned. Samples were subjected to antigen retrieval with sodium metaperiodate and subsequently incubated with primary anti-FITC antibodies (mouse monoclonal IgG, Abcam, ab10257, 1 ug/ml, diluted in 0.2% Aurion BSA (AURION, Wageningen, the Netherlands) in PBS pH 7.6.) followed by detection with species-specific secondary antibody-gold conjugates (EM goat anti-mouse IgG 15 nm Gold, BBInternational, Cardiff, UK). The sections were stained with uranyl acetate (0.5%, Laurylab, Saint Fons, France) and lead citrate (3%, Laurylab). Rabbit ChP were examined in a FEI Technai Biotwin 120kv TEM operated at 100 kV accelerating voltage. Images were recorded with side-mounted Olympus Veleta camera with a resolution of 2048 × 2048 pixels (FEI, Hillsboro, OR, USA).