Fluoromount g media

Cryosections collected in slides were thawed and hydrated in PBS for 15 minutes at room temperature (RT). For antibody staining, sections were permeabilized with PBS containing 0.1% Tween20 (PBT) for 15 min at RT, blocked with 10% donkey serum (Jackson ImmunoResearch) in PBT and incubated with primary antibody, diluted in blocking solution, overnight at 4°C. Primary antibody was washed 3 times with PBT and detected using Alexa conjugated secondary antibodies (Invitrogen, Molecular Probes). Secondary antibody was washed 6 times with PBT. For nuclei staining, slides were incubated for 5min in 300nM DAPI (4',6-diamidino-2-phenylindole) in PBS at RT, rinsed in PBS and mounted with Fluoromount G media (17984-25, Electron Microscopy Sciences) and micro cover glass No. 2 (48382-128, VWR). Primary antibodies used were as follows: rabbit anti-MANF (1:300; Sigma, SAB3500384); rat anti-CD68 (1:100, AbD Serotec (FA-11)); goat anti-IBA1 (1:100; Novus Biologicals, NB1001028SS); and goat anti-GFP (1:800; NB 100-1770, Novus Biologicals).

Cryosections collected on slides were thawed, permeabilized with PBS containing 0.1% Tween20 (PBT) for 15 min at RT, blocked with 10% donkey serum (Jackson ImmunoResearch) in PBT and incubated with primary antibody, diluted in blocking solution, overnight at 4°C. Primary antibody was washed 3x with PBT and detected using Alexa conjugated secondary antibodies (Invitrogen, Molecular Probes). Secondary antibody was washed 6x with PBT. Nuclei were stained for 5min with 300nM DAPI (4’,6-diamidino-2-phenylindole) in PBS at RT. Neutral lipids were stained for 5 min with 50μg/ml Bodipy 493/503 (Invitrogen, D3922) in PBS at RT. Slides were rinsed in PBS and mounted with Fluoromount G media (17984–25, Electron Microscopy Sciences) and micro cover glass No. 2 (48382–128, VWR). Staining of pJNK, γh2Ax and cCasp3 included an additional step of permeabilization involving 2 min incubation in boiling 10mM Citrate buffer. For information on primary antibodies used see Supplementary Table 3.