Fitc conjugated anti rabbit igg

The human intestinal epithelial cell line CACO-2 (ATCC) was grown in vitro as per recommendations. Expression of the tight junction protein ZO-1 was measured by immunofluorescence using a purified anti-ZO-1 antibody (Life Technologies) as the primary antibody and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories) as the secondary antibody. Expression of ZO-1 was observed using confocal microscopy (Leica DM2500, LAS-AF) and the mean florescence intensity of ZO-1 expression was calculated using image J software.

Western blotting and immunohistochemical (fluorescence) staining were performed as previously described [37 (link), 38 (link)]. The primary antibodies used in this study were AHR (RPT9), HDAC8 (GeneTex), RB1, p53, Cyclin D1, PCNA, ARNT1, CYP1B1 and E2F1 (1:200), actin polyclonal antibodies (1:5000 dilution; Sigma–Aldrich), HA and GFP monoclonal antibodies (1:200 dilution; Upstate, NY, USA), FITC-conjugated anti-rabbit IgG, rhodamine-conjugated anti-mouse IgG, alkaline phosphatase-conjugated anti-rabbit IgG antibody (1:500 dilution; Jackson ImmunoResearch Laboratories), and Ki67 goat polyclonal antibody (1:200 dilution; Santa Cruz Biotechnology) [28 (link)]. All of the experiments were repeated at least three times, and staining with relevant control antibodies was performed in parallel and in all cases no signals were seen (for example, Supplementary Figure S1).

Lung tissue specimens obtained from each tumour-resected mouse were fixed with 4% formalin and embedded in paraffin. For histological comparison, 6-μm-thick tissue sections were made and stained with hematoxylin and eosin (H&E). For immunofluorescence staining, fixed tissue sections were initially immersed in boiling sodium citrate buffer (0.01 M sodium citrate buffer, pH 6.0) for 30 min. Lung tissue sections were blocked with 5% nonfat milk, and incubated with anti-FSP-1 antibody (1:200 dilution; Millipore; catalogue number: 07-2274), FITC-conjugated anti-CD206 antibody (1:200 dilution; Biolegend; catalogue number: 141703) or PE-conjugated anti-IL-25 antibody (1:100; R&D; catalogue number: IC12991P) in 1% nonfat milk for 1 h at room temperature. Sections were then washed with PBS containing 0.1% Tween 20. To detect primary antibodies, some sections were incubated with FITC–conjugated anti-rabbit-IgG (1:200; Jackson Immunoresearch, West Grove, PA; catalogue number: 111-097-103) for FSP-1. 4′,6-Diamidino-2-phenylindole dihydrochloride (1 μg ml⁻¹; Sigma-Aldrich) was used to stain the nuclei. Fluorescence microscopy evaluation of immunostained tissue sections was performed using a Zeiss Axiovert 200 M microscope (Carl Zeiss, Heidelberg, Germany). Images were captured with a digital camera (Orca ER, Hamamatsu) and processed using Axiovision 4.6.3 (Carl Zeiss).

Immunofluorescence analysis was done on the cultured cells according to the protocol described by Wittmann et al. [15] (link). Briefly, M14 and ARO cells were grown on glass coverslips, washed twice with phosphate-buffered saline (PBS) and fixed with cold methanol for 30 minutes. Subsequently, the cells were permeabilized with PBS containing 0,2% TritonX-100 (PBST) for 30 minutes and blocked with PBST containing 2% BSA (Serva). Immunostaining was performed using a TPX2 (D2R5C) XP monoclonal antibody (Cell Signaling Technology) (1∶500 diluted in PBST containing 1% BSA) for 1 hours. Following the washes with PBS, the cells were incubated for 1 h in the dark with a secondary antibody FITC-conjugated anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) (1∶200 diluted in PBST containing 1% BSA). The nuclei were finally counterstained with diamidinophenylindole 50 ng/ml for 1 minute. After washing with PBS, glass microscopy slides, mounted with Vectashield, were examined by microscope (Leica AF6000 Modular Systems) with 63× oil immersion objective.

(i) First antibodies. The following first antibodies were used: Mouse anti-phospho-H2AX (Ser139) (#05-636), mouse anti-β-actin (#A5441) from MilliporeSigma rabbit anti-phospho- RPA32 (Thr21) (#AP1040), rabbit anti-phospho-DNA-PKcs (Ser2056) (#AP0621) and, rabbit anti-Pol κ (#A6122) from Abclonal, rabbit anti-phospho-ATR (Thr1989) (#GTX128145) and rabbit anti-polymerase η (#GTX109938) from GeneTex, mouse anti-AAV2 Rep protein (#03-61069; #03-61073) and rabbit anti-AAV2 capsid proteins (#03-61084) from ARP. Rat anti-MAAP was generated by immunizing rats with purified GST-MAAP, following a previously published protocol (86 (link)). Rabbit anti-AAP antisera were made by immunization of 2 rabbits with a peptide antigen, Cys-RSTSSRTSSARRIKDASRR (87 (link)), at Biomatik Co.
(ii) Secondary antibodies. The following secondary antibodies were used: DyLight 800 conjugated anti-rabbit IgG (#5151S) and DyLight 800 conjugated anti-mouse IgG (#5257S) (Cell Signaling, DyLight 800 conjugated anti-rat IgG (#SA5-10024) ThermoFisher, FITC conjugated anti-mouse IgG (#115-095-003), FITC conjugated anti-rabbit IgG (#111-095-003), Alexa Fluor 488 conjugated goat anti-mouse IgG (#115-545-062), and Alexa Fluor 594 conjugated goat anti-mouse IgG (#115-585-146) (Jackson ImmunoResearch).

The cells were grown onto poly (D-lysine)-coated cover glasses in a 24-well plate. After treatment, the cells were fixed for 20 min using cooled absolute ethyl alcohol, and then incubated with PBS containing 0.2% Triton X-100 for 30 min. After blocking with 5% non-fat milk in PBS for 1 h, the cells were then incubated with antibodies against GRP78 (1:500; Santa Cruz, sc-1050) or LC3 antibody (1: 400; MBL, PD 014) at 4°C for 24 h. The cells were sequentially incubated with Cy3-conjugated anti-goat IgG (1:400; Jackson ImmunoResearch, 705-166-147) and FITC-conjugated anti-rabbit IgG (1:400; Jackson ImmunoResearch, 111-095-003) for 2 h. Afterwards, the cells were incubated with 0.5 μg/ml 4,6-diamidino-2- phenylindole (DAPI, Sigma) for 10 min. Then the cells were mounted on slides and images of fluorescence were acquired using a laser confocal microscope (Carl Zeiss Microimage Inc., Thornwood, NY).