Nbp1 19371

The rats were ethically euthanized at selected time points. For histological observation, enucleated eyes (n = 6 at each time point) were fixed in 10% formaldehyde, dehydrated with gradient alcohol, and embedded in paraffin. Sections of 5 μm were made and deparaffinized before treatment with hematoxylin and eosin (H and E). For immunofluorescence, the eyeballs were fixed in 4% paraformaldehyde (PFA) after enucleation overnight, dehydrated in 30% sucrose, and embedded in OCT compound. Serial 5 μm sections were fixed with 4% PFA for 15 min and permeabilized with 0.1% Triton-X-100 for 15 min at room temperature (RT). Sections were blocked with 5% bovine serum albumin and incubated with primary antibodies against CD4 (1:50, NBP1-19371, Novus Biologicals, Centennial, CO, USA) and IL-17A (1:50, sc-374218, Santa Cruz, CA, USA) at 4 °C overnight. Alexa Fluor 488 donkey anti-rabbit IgG (1:1000, ab150073, Abcam, Cambridge, UK) and Alexa Fluor 647 goat anti-mouse IgG H&L (1:1000, ab150115, Abcam, Cambridge, UK) were used as secondary antibodies at 37 °C for 1 h. Nuclei were stained with 4,6-diamino-2-phenylindole (DAPI) for 10 min at RT. The specimens were washed in phosphate-buffered saline, mounted onto coverslips, and analyzed using confocal microscopy (Carl Zeiss, Oberkochen, Germany).

Freshly harvested tissues were fixed with phosphate-buffered formalin for a minimum of 48 h and then embedded in paraffin wax for histological analysis. The slides were stained with hematoxylin and eosin stain (H&E), and the images were captured as previously published [12 (link)]. Four to six images per section of each lung were scored blindly. For each sample, histologic evidence of pathology was classified in terms of the presence of infiltrated immune cells, lipid droplets, and foamy macrophages and was graded on a 7-point scale ranging from 0 to 6. Auramine-rhodamine (AR) staining of the lung and adipose tissue sections was performed, and the images were captured [4 (link)]. Four to six images per section of each lung were quantitated for fluorescence intensity by Image J. Immunohistochemical analysis (IHC) was performed on the formalin-fixed lung using CD4 specific rabbit polyclonal antibody (#NBP1-19371, Novus Biologicals), CD8 specific rabbit polyclonal antibody (#NBP2-29475, Novus Biologicals), F4/80-specific rabbit polyclonal (#ab100790, Abcam), and IFNγ-specific rabbit polyclonal antibody (#BS-0480R, Bioss Antibodies) as described earlier [13 (link)]. We analyzed the antibody-stained slides with semi-quantitative scoring system and graded them on a 7-point scale ranging from 0 to 6.

Lung and tumor tissues from experimental mice were harvested and embedded in OCT compound (Tissue-Tek, Torrance, CA). Cryosections (4-5 μm) were fixed in 100% acetone, followed by blocking with 3% bovine serum albumin (GenDEPOT, Barker, TX, USA) solution. The sections were stained with anti-CD3 (ab16669, Abcam), anti-CD4 (NBP1-19371, Novus Biologicals, CO, USA), anti-CD8 (ab4055, Abcam), anti-CD11c (ab11029, Abcam), anti-F4/80 (ab6640, Abcam), fibronectin (ab2413, Abcam) and vimentin (ab193555, Abcam) overnight at 4 °C. On the next day, sections were incubated with an Alexa Fluor^® 488- and 647-labeled secondary antibody (Abcam). Images of issues were acquired using a LSM880 confocal microscope (Zeiss, Jena, Germany) using the Zen 2.3 software (Zeiss) for image processing and quantitative analysis. The arithmetic mean of the optical intensities of the target proteins were measured in individual slide and normalized to those of DAPI (4′,6-diamidino-2-phenylindole) using the Zen 2.3 software (Zeiss, Jena, Germany). All experiments were performed in three randomly selected groups.