*2.3. Immunofluorescence and Differential Interference Contrast Imaging of Lung NETs*

Formalin fixed paraffin embedded mouse lung sections (3 μm) were rehydrated and heat induced antigen retrieval performed in 0.01 M citrate buffer pH 6.0 for 20 min. Sections were blocked with 1% normal swine serum (NSS, DAKO, Carpinteria, CA, USA) and incubated with primary antibody #1, rat anti-mouse CD41 (MWReg30, ab33661, Abcam, Cambridge, MA, USA), 1:10 diluted in 1% NSS/PBS overnight at 4 °C. Sections were then incubated with goat anti-rat Alexa Fluor® 488 1:50 (Abcam) in PBS for 4 h followed by incubation with primary antibody #2, rabbit anti-myeloperoxidase (ab45977, Abcam) 1:10 diluted in PBS overnight at 4 °C. Sections were then incubated with chicken anti-rabbit Alexa Fluor® 647 (Invitrogen, Life Technologies, Grand Island, NY, USA) 1:50 in PBS for 4 h followed by incubation with primary antibody #3, mouse anti-histone H2A (L88A6, Cell Signaling, Danvers, MA, USA) 1:200 in PBS overnight at 4 °C, and then finally incubated with goat anti-mouse IgG1 Alexa Fluor® 594 (Invitrogen) 1:50 in PBS for 4 h. Nuclear counterstain was performed with DAPI (Invitrogen) 1:500 for 5 min and sections mounted with Slow Fade Gold (Invitrogen). Negative controls were run in parallel with nonspecific IgG or specific isotype. Confocal microscopy was performed with a Leica TCS SP2 laser scanning confocal microscopy system of the VCU Department of Anatomy and Neurobiology Microscope Facility. Separate images of optical sections were acquired with filters for Alexa Fluor® (AF) 488, 594, 647 and DAPI. Images were assembled with ImageJ software.

Isolation of total RNA and real-time QPCR analyses were performed as described previously [12]. Primers used for QPCR are listed in Table 1.


**Table 1.** Murine primers used for Quantitative PCR (QPCR).
