Alexa fluor 488 green

Sections from day 2 scarified mouse lung were deparaffinized and rehydrated with Xylene and different concentration ethanol. The slides were permeabilized with 0.1% Triton X-100 in PBS. After washing slides with PBS three times, sections were blocked with 5% FBS in 0.1% PBS, and then incubated overnight with first antibody, anti-F4/80 (Abcam) and anti-KC (R&D Systems, Bio-Techne, Minneapolis, MI, USA) (1:100 dilution) at 4 °C. After incubation, slides were washed with PBS twice and tagged with secondary antibody Alexa Fluor 488 (green) or Alexa Fluor 647 (red) (Abcam)for 1 h at room temperature. Slides were mounted with DAPI and imaged by fluorescence microscopy.

NET identification in tissue samples was performed by immunofluorescence staining. An anti-NE antibody (MAB91671) was purchased from R&D Systems (Minneapolis, USA) (1:50 dilution). An anti-H3Cit antibody (ab5103) was obtained from Abcam (Cambridge, UK) (1:50 dilution). The NE/H3Cit pair was researched in paraffin-embedded, 3-μm-thick sections. The slides were incubated with the primary antibodies at 4°C overnight after blocking with goat serum. Then, the sections were incubated with secondary antibodies (Alexa Fluor 488, green; and Alexa Fluor 647, red) (Abcam, Cambridge, UK) for 1 h at room temperature. DAPI was used for nuclear staining (ZSGB Biotech, Beijing, China). Finally, the slides were analyzed with a confocal laser scanning microscope (TCS-SP5; Leica, Wetzlar, Germany). For each specimen, the position of tumor and paratumor tissue was determined according to the results of H&E staining. Next, the average numbers of NE and H3Cit double-positive cells of tumor and paratumor tissues were calculated by two researchers counting five random 630x microscopic fields, respectively.

NSCs culture medium was under support from Chi Scientific (Jiangsu, China). Anti-NGF, anti-LC3-II, anti-Beclin1, anti-P62, anti-NeuN, donkey anti-rabbit IgG and anti-mouse IgG with Alexa Fluor 594 (red) and Alexa-Fluor 488 (green) were supplied by Abcam (Cambridge, Britain). DAPI (4′,6-diamidino-2-phenylindole), a fluorescent agent used for nuclear staining, was purchased from Sigma-Aldrich (St. Louis, MO, USA).