Nis elements br analysis software

The two ELISA-positive samples selected from late and fast feathering chicken groups were also evaluated with Immunofluorescence assays (IFA) and Western blotting following our previously described method with ALV-J envelope protein specific monoclonal antibody JE9 (kindly provided by Dr. Kun Qian, Yangzhou University) (Venugopal et al., 1997 (link); Dai et al., 2016a (link)). IFA was performed using the FITC-labeled anti-mouse IgG (Sigma, USA) and analyzed by fluorescence microscope using NIS-Elements BR analysis software (Nikon, Japan). IRDye 800-conjugated anti-mouse IgG or IRDye 700DX-conjugated anti-rabbit IgG (1:10,000; Rockland Immunochemicals, USA) were as the secondary antibody in western blot analysis, and analyzed with an Odyssey infrared imaging system (LI-COR Biosciences, USA).

Double‐label immunofluorescence staining and image analysis were performed as previously described.
⁷ (link) Sections were immersed in 5% goat serum for 1 h and then incubated overnight at 4°C with primary antibodies, including anti‐connexin 43 (Cx43, Abcam, ab66151, 1:200), anti‐glial fibrillary acidic protein (GFAP, Millipore, MAB360, 1:600). Secondary antibodies conjugated with Alexa Fluor 488 (SouthernBiotech, 1036–02, 1:400) and 594 (SouthernBiotech, 4030–03, 1:400) were diluted in blocking buffer and incubated for 2 h at 37°C. The brain slices were attached to coverslips and mounted with VECTASHIELD Mounting Medium (Vector Laboratories, USA) containing correct dilution of DAPI (SouthernBiotech, USA). All slices were viewed using a fluorescent microscopy (Nikon, Japan). Non‐overlapping regions (3 for right peri‐hippocampal cortex and bilateral Cornus Amonis area 1 (CA1); 2 for bilateral dentate gyrus (DG)) were randomly collected. All images were analyzed with NIS‐Elements BR Analysis software (Nikon, Japan). The positive expression of GFAP/Cx43 was represented with integrated optical density (IOD).
³⁵ (link) Data acquisitions and analyses were performed in blind conditions of the experiments.

DNA was extracted from the spleens of the clinic samples using a commercial kit (Omega, USA). Specific primers were employed to differentiate between ALV-J, ALV-A/B, and other suspected viruses including Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) (Lai et al., 2011 (link)). According to a previously described method (Li et al., 2013 (link)), chicken plasma was used for virus isolation by inoculation into DF1 cells. DF-1 cells are only susceptible to exogenous ALV virions (Maas et al., 2006 (link)). Infected DF1 cell culture supernatants from clinical samples were tested for ALV group-specific antigen (p27) using the ALV Antigen Test Kit^R (IDEXX, USA) according to the manufacturer’s instructions. ALV-J infection was further confirmed by immunofluorescence using standard techniques (Venugopal et al., 1997 (link)). Infected cell images were collected using NIS-Elements BR analysis software (Nikon).
Liver tissue samples fixed in 10% buffered formalin were stained with hemotoxylin and eosin (HE) and examined histopathologically (Cheng et al., 2010 (link)). Immunohistochemical staining was used for further diagnoses with JE9 monoclonal antibody (kindly provided by Dr. Kun Qian, Yangzhou University). Binding of the JE9 antibody was detected using anti-mouse-HRP (Zhongshan Goldenbridge, Beijing, China).

The clinical symptoms of 140-day-old spontaneous infection female White Recessive Rock (WRR) chickens were as follows: depression and hemorrhages in the skin of the phalanges and feather follicles. In total, 23 tumor spleen samples and 23 healthy spleen samples from chickens were collected independently. The procedures for virus isolation were performed in DF-1 cells as described previously45 (link). After the infected DF-l cells were cultured for 7 days, the supernatants of these cells were harvested and tested for ALV group-specific antigen (p27) using an antigen-capture ELISA kit (IDEXX Laboratories, USA). PCR was used to test genomic DNA from cultured DF-1 cells. Previously published specific primers (Supplementary Table S5) were synthesized and employed to detect different subgroups of exogenous ALVs46 (link) and other avian tumor viruses, including MDV47 and REV48 . ALV-J infection was further confirmed by IFA using standard techniques49 (link). The images of infected cells were collected by fluorescence microscope (Nikon, Japan) using NIS-Elements BR analysis software (Nikon). Finally, spleens from the two groups (ALV-J-infected group: WRR⁺; uninfected group: WRR⁻) were subjected to Illumina deep sequencing.

Neonate mice were immediately sacrificed after birth by neck removal. Lung tissue was perfused with PBS and fixed in 4% buffered formalin overnight, followed by dehydration, embedding, and sectioning following standard protocols. Sections (5 μm) were mounted on slides for H&E staining. Aerated lung areas and alveolar septal thickness were measured in H&E stained sections using Nikon NIS-Elements BR Analysis software. Septal thickness was measured at all points in each visual field, and five visual fields for each animal were used for the measurement. According to the manufacturer's instructions, sections were also stained with Periodic Acid Schiff stain (PAS, Sigma-Aldrich, St. Louis, MO, #395) to visualize intracellular glycogen. For immunohistochemical staining, tissue sections were deparaffinized and then rehydrated in graded ethanol from 100% to 75%. For antigen retrieval, sections were incubated with 0.1% proteinase K (#19131, Qiagen) for 15 min at room temperature. After washing with PBS, sections were subjected to UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit according to the manufacturer's instructions (MXB KIT-9720, MXB Biotechnologies, China). Brown signals were visualized using DAB (MXB 0031, MXB Biotechnologies, China). Slides without primary antibodies were used as negative controls.

Those samples that tested positive for infection by ELISA were also tested by IFA. Briefly, the plasma was used to inoculate a monolayered 24-well plate. After incubation for 2 h, the supernatant was removed, and a 1% FBS cell maintenance solution was added to prolong the culture for 4–5 days. After fixing and sealing the cells, the ALV-specific monoclonal antibody JE9 (kindly provided by Dr. Aijian Qin, Yangzhou University, Yangzhou, China) as the primary antibody and FITC-labeled antimouse IgG (Bioss, Beijing, China) were used. Cell morphology was observed under a fluorescence microscope, with images recorded using NIS-Elements BR analysis software (Nikon, Japan).

Immunohistochemistry was performed in 4 μm formalin-fixed kidney slides. Sections were deparaffinised and endogenous peroxidase was blocked with 2% hydrogen peroxide/methanol. Pronase, Proteinase K and heat mediated sodium citrate antigen retrieval was performed for NIMP, F4/80 and C3 staining respectively. Afterwards endogenous avidin and biotin were blocked. Blocking solution and primary antibody was added overnight (NIMP, F4/80 and C3 from Abcam). After the addition of secondary biotinylated antibody and ABC, sections were developed with DAB, counterstain with Mayer’s haematoxylin and mounted with Pertex. NIMP+ cells were manually counted in at least 15 random glomeruli per section. F4/80 and C3 morphometric image analysis was performed at 400 × using Nikon Eclipse Upright microscope and NIS-Elements BR Analysis software from Nikon. At least 10 random glomeruli were analysed per kidney and data is expressed as percentage of positive staining per glomeruli.