Mouse il 1β

The eyes were processed, sectioned, and stained with hematoxylin and eosin as previously described [6 (link)]. Sections were also subjected to immunohistochemical staining with antibodies against mouse IL-1β, TNF-α, or MCP-1 (Abcam, Cambridge, MA, USA). Additionally, sections were subjected to immunofluorescence staining with DAPI (4′,6-diamidino-2-phenylindole) for DNA and the antibody against mouse p65 (Abcam, Cambridge, MA, USA).

The cell cultures were fixed with 4% paraformaldehyde in cytoskeleton buffer with sucrose (CBS) (Posada-Duque et al., 2017 (link)). Autofluorescence was eliminated using 50 mM ammonium chloride (NH4Cl) for 10 min. The cells were permeabilized with 0.2% Triton. X-100 prepared in CBS and then treated with a blocking solution (2.5% FBS in CBS). The cultures were incubated overnight at 4°C with mouse primary antibodies against mouse CLDN5 (Invitrogen, 1:750), mouse GFAP (Sigma-Aldrich, 1:750), rabbit BACE1 (Abcam, 1:500) and mouse IL-1β (Abcam, 1:500). Subsequently, the cultures were incubated for 1 hour with Alexa 594- or Alexa 488-tagged secondary antibodies (Molecular Probes, 1:500), and the nuclei and cytoskeleton were stained with Hoechst 33258 (Invitrogen, 1:5000) and phalloidin conjugated with Alexa 594 or Alexa 488 (1:500, Molecular Probes). Then, serial washes with PBS (phosphate-buffered saline) were performed, and the coverslips with the immunolabeled cells were fixed to slides with FluorSave. The cells were observed under an Olympus IX 81 epifluorescence microscope, and the images were captured with an oil immersion objective (60X, NA 1.42) and then processed.

Paired antibodies and standard recombinant human IL-1β (RD), IL-18 (Abcam), caspase-1 (Abcam) and mouse IL-1β (Abcam) and caspase-1 (Novus) were used to determine cytokine concentrations according to manufacturer’s instructions. A microplate reader (Bio-Rad) was used to detect the signals at 450 nm.