Ab21176

For immunofluorescence staining, antigen retrieval of deparaffinized tissue sections was performed by microwave on the middle for 20 min. After being blocked with 5% donkey serum (Solarbio, Beijing, China) for 1 h at room temperature, the sections were incubated with primary antibodies at 4°C overnight, followed by incubation with anti-rabbit Alexa Fluor 488 (A21206; Life Technologies, Carlsbad, USA) or anti-goat Alexa Fluor 594 (A11058; Life Technologies) conjugated secondary antibodies. DAPI (Life Technologies) staining was performed for nuclear counterstaining. Fluorescent images were visualized by using a laser-scanning confocal microscope system (OLYMPUS, Tokyo, Japan). The following primary antibodies were used: goat polyclonal anti
-MCP-1 antibody (sc-1785; Santa Cruz Biotechnology, Dallas, USA) and rabbit polyclonal anti-Firefly Luciferase antibody (ab21176; Abcam, Cambridge, UK).

As we have previously described [14 (link)], mice were killed at the indicated time points after challenge and xenograft tissues were trisected for histology and fluorescence staining. Samples for histological analysis were fixed in neutral buffered 4% paraformaldehyde (PFA), paraffin embedded, and sections were cut at a thickness of 5 μm and stained with hematoxylin and eosin (H&E). Fresh xenograft tissue for fluorescence staining was fixed in 2.5% PFA overnight at room temperature, incubated with 15% (w/v) sucrose for 12 hours at 4°C and frozen in Tissue-Tek® (EMS, Hatfield, PA) embedding medium. Serial 10 μm cryosections were stained with phalloidin (Sigma) and 4’, 6’-diamidino-2-phenylindole (DAPI) (Sigma). For immunofluorescence staining primary antibodies were anti-luciferase antibody (ab21176 Abcam, Cambridge, UK) and alexa fluor 594 Donkey anti rabbit (A21207 Invitrogen, Carlsbad, CA) were used as secondary antibodies. Sections were mounted with VectaShield (Vector Laboratories,Burlingame, CA) and imaged with an Axio Imager M1 upright light microscope (Zeiss, Germany) coupled to a MR3 CCD camera system (ZEN 2012).

Proteins were extracted using Bicine/CHAPS lysis buffer. Lysates were then separated via gel electrophoresis (Bio-Rad) and transferred to PVDF membranes using standard protocols. MYC and FLuc protein levels were detected using an anti-MYC (ab32072, Abcam)^{51 (link)} or anti-FLuc (ab21176, Abcam)^{52 (link)} antibody, respectively. The blot was imaged using a LI-COR scanner and analyzed by ImageJ.
Paraffin-embedded tumor sections were deparaffinized by successive incubation in xylene, 95% ethanol, 90% ethanol, and 70% ethanol, followed by PBS. Epitopes were unmasked by steaming in DAKO antigen retrieval solution for 45 min and then rinsed twice in PBS. The sections were blocked using DAKO blocking solution, and then immunostained overnight at 4 °C using primary antibodies (MYC, 1:150, Epitomics). Sections were then washed with PBS and incubated with biotinylated anti-rabbit IgG (1:300, Vector Labs) for 30 min at room temperature, then with the ABC kit (Vector Labs) for 30 min at room temperature. Sections were developed using 3,3′-diaminobenzidine (DAB, Vector Labs), counterstained with hematoxylin, and mounted with Permount. Images were obtained on a Philips Ultrafast Scanner.

Transfected cells were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.1% Triton X-100 for 5 min. Samples were washed three times with 1% PBST, followed by blocking with protein block serum-free (Dako) for 1 h and incubation with ANTI-FLAG M2 (1:500; F1804, SIGMA)/anti-firefly luciferase antibody (1:200; ab21176, Abcam) overnight at 4 °C. Following three washes with PBST, the samples were incubated with Alexa Fluor 568-conjugated goat anti-mouse IgG antibodies (1:500; Invitrogen)/Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies (1:500; Invitrogen) and DAPI (1:500; Dojindo) for 1 hour at RT to visualize the antigens and nuclei. These were mounted with Ultramount aqueous permanent mounting medium (DakoCytomation) and visualized under a confocal fluorescent microscope (LSM700, Zeiss).