Mouse monoclonal anti gfp b 2

Immunocytochemistry was performed using WT HepaRG‐derived HLCs to exam protein expression, because transgenic cells express fluorescent. Cells were fixed with PBS containing 2% (w/v) paraformaldehyde (PFA) for 5 min and washed with PBS. Permeabilization was performed with PBS containing 0.1% Triton X for 10 min, followed by washing with PBS. After incubation in PBS containing 2% skim milk for 30 min, cells were incubated with rabbit polyclonal anti‐human CYP3A4 antibody (Enzo Life Sciences), mouse monoclonal anti‐human cytokeratin 19 antibody (CK19; DakoCytomation), mouse monoclonal anti‐human albumin (ALB; Sigma‐Aldrich), mouse monoclonal anti‐keratin K8/K18 (CK8/18; PROGEN), hepatocyte nuclear factor 4 alpha (HNF4A), and mouse monoclonal anti‐GFP (B2; Santa Cruz Biotechnology), diluted 1:500 in PBS with 2% skim milk for 1 hours at room temperature. Then, samples were incubated with the secondary antibodies Alexa Fluor® 488‐conjugated rabbit IgG and Alexa Fluor® 546‐conjugated mouse IgG (Invitrogen) diluted 1:500 in PBS with 2% skim milk for 1 hours at room temperature. Samples were washed three times with PBS containing 0.05% Tween‐20 for 10 minutes. DNA was stained with Hoechst 33 258 solution (Merck).

Cells were grown to 70% of confluence and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed 2× in PBS and stored in 70% EtOH at 4 °C. Cells were rehydrated in PBS and permeabilized in 0.5% triton X-100 in PBS for 10 min at room temperature. A blocking step was performed with 2% BSA in PBS for 30 min at room temperature. The primary antibody (mouse monoclonal anti-GFP (B-2); Santa Cruz)was diluted 1:500 in PBS and incubated for 1 h at room temperature, washed three times with PBS, and incubated with secondary antibody (Alexa 488 conjugated Goat Anti-mouse IgG (H + L); ThermoFisher), which was diluted 1:1000 in PBS. Cells were imaged on a Zeiss AxioObserver microscope with a Hxp120V light source, an Axiocam 702 mono camera and a Plan-Neofluar 40 × 0.75 NA objective.

CpG oligodeoxynucleotide-1826 (CpG DNA) and biotin-CpG DNA were purchased from TIB Molbiol (Germany). R848 and LPS (Escherichia coli O111:B4) were from Enzo Life Sciences (USA) and Sigma-Aldrich (USA), respectively. Rat monoclonal anti-HA (3F10) was purchased from Roche (USA). Mouse monoclonal anti-GFP (B-2) was purchased from Santa Cruz (USA). Mouse monoclonal anti-myc (9B11), rabbit monoclonal anti-MyD88 (D80F5), mouse monoclonal anti-phospho-Iκβ (5A5), and anti-Iκβ were obtained from Cell Signaling Technology (USA). Rat anti-LAMP1 (1D4B) was obtained from BD Biosciences (USA). Rabbit anti-actin was obtained from Bethyl Laboratories (USA). Rabbit anti-GFP and rabbit anti-myc antibodies were made in our laboratory by immunizing rabbits with purified GFP protein and Myc epitope tag peptide (EQKLISEEDL), respectively.