Permafluor medium

Eyes were cleaned up from muscles and incubated during 2 min in Pursept-AXpress (Thermo Fisher Scientific, Waltham, MA, USA) for disinfection. The anterior segment was cut along the limbus to remove the cornea, lens and vitreous. The retina was carefully removed from the eyecup and placed into CO₂ independent medium (Life Technologies, Carlsbad, CA, USA). A 1 cm² piece was cut and either immediately flat-mounted in Permafluor medium (Thermo Fisher Scientific) or included in 2–4% low melting agarose dissolved in a pre-warmed (+37 °C) custom modified Neurobasal A medium without any photosensitizer such as phenol red, riboflavin, folic acid and aromatic amino acids (modified NBA, Life Technologies) for retinal sectioning. Transversal slices of 100 µm thickness were cut using a vibrating microtome (Leica, Wetzlar, Germany) in modified NBA medium and then mounted in Permafluor medium (Thermo Fisher Scientific). Images were acquired without further delays.

Optimal cutting temperature compound embedded frozen tumor tissues sections (5 µm) were incubated with CD206, iNOS, Ly6G, and p-Smad3 antibodies overnight at 4 °C, followed by incubation with Alexa Fluor 546-conjugated or Alexa Four 488-conjugated secondary antibodies (1:1,000; Invitrogen) in staining buffer (eBioscience) for 2 h at room temperature. After washing with PBS, the sections were stained with FITC-conjugated Ly6G or PE-conjugated CD16b overnight at 4 °C. Nuclei were stained with Hoechst 33342 (Invitrogen) for 5 min in PBS, then mounted with PermaFluor medium (Thermo Fisher Scientific)^{52 (link)}. Images and z-stack scanning were acquired by Zeiss Axio Observer.Z1 and LSM 880 fluorescence microscopes, respectively, and analyzed with ZEN lite image analysis software 2.4.

Optimal cutting temperature (OCT) compound (Sakura, Ref-4583)–embedded tumor tissues (5 μm thickness) were used for immunohistochemical staining. After OCT removal and blocking, sections were incubated with primary antibody overnight at 4°C. For nonconjugated primary antibody, sections were incubated with Alexa Fluor 488/546–conjugated secondary antibody (Thermo Fisher Scientific, 1:1000; α-Ms: F-2761, A-11003, α-Rb: A-11008, A-11010) in staining buffer for 2 hours at room temperature (eBioscience, 00-4222-26). Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, H1399) and then mounted with PermaFluor medium (Thermo Fisher Scientific, TA-030-FM). Images were captured with a Zeiss fluorescence microscope and analyzed with ZEN image analysis software.