Teleostean gelatin

Protozoa were washed in PBS and fixed with freshly prepared 2% formaldehyde diluted in PBS, for 1 h. After fixation, cells were adhered to poly-l-lysine-coated microscope coverslips and permeabilized with 4% Nonidet P-40 (NP-40) diluted in PBS for 45 min. Slides were incubated in blocking solution containing 1.5% bovine serum albumin (BSA), 0.5% teleostean gelatin (Sigma Aldrich), and 0.02% Tween 20 diluted in PBS. Next, slides were incubated for 1 h with antibody produced against the symbiont porin^{54 (link)} diluted 1:10 in blocking solution. After that step, the cells were washed with PBS and incubated for 45 min with Alexa488-conjugated anti-mouse IgG (Molecular Probes, USA) diluted 1:200 in blocking solution. Slides were mounted using the anti-fading reagent ProLong Gold containing 5 μg/mL of DAPI (4′,6-diamidino-2-phenylindole, MolecularProbes). Serial image stacks (0.36-μm Z-increment) were collected at 64× (oil immersion 1.4 NA) on an Elyra PS.1 microscope (Carl Zeiss) and three-dimensional projections were obtained on the Zen Black program (Carl Zeiss).

Formalin-fixed paraffin-embedded human tissue sections were deparaffinized by incubation in xylene, rehydrated by a graded series of ethanol and rinsed in distilled water for 5 min. For antigen retrieval, the slides were steamed in 0.01 M sodium citrate buffer, pH 6.0 at 100 °C for 20 min, followed by three washes in TBS. Endogenous peroxidases were quenched with 0.3% hydrogen peroxide for 20 min. Sections were blocked with a solution composed of TBS, 0.1% Triton X-100, 1% BSA and 0.2% teleostean gelatin (Sigma) for 20 min, and incubated overnight at 4 °C with a rabbit anti-5-LOX antibody (1:50; 610695, BD Pharmingen) diluted in the blocking solution. After incubation, the sections were washed in TBS, incubated with a biotinylated rabbit anti-mouse antibody (1:500, Jackson Immuno Research) for 1 h, washed in TBS, and incubated for 1 h in a peroxidase-avidin complex solution (Vectastain Elite ABC kit; Vector Laboratories). The peroxidase activity of immune complexes was revealed with a solution of TBS containing 0.25 mg ml⁻¹ 3,3 diaminobenzidine (Vector Laboratories), 0.01% H₂O₂, and 0.04% NiCl₂. Sections were mounted with Neo-Mount (Merck).

For immunocytochemical analysis of BV-2 cells and mouse neurospheres from FoxO1/3/4^fl/fl mice, fixed cells were washed in TBS (0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5), then blocked with a solution composed of TBS, 1% BSA and 0.2% Teleostean gelatin (Sigma) (fish gelatin buffer, FSGB). The same solution was used during the incubations with antibodies. Primary antibodies were applied overnight at 4 °C. Fluorochrome-conjugated species-specific secondary antibodies were used for immunodetection. The following antibodies and final dilutions were used. Primary antibodies: rabbit anti-CysLTR1 (1:500, SP4109P, Acris), chicken anti-GFP (1:1,000; GFP1020, Aves Labs) and rabbit anti-GPR17 (1:400, 10136, Cayman).Secondary antibodies: donkey anti-rabbit Alexa 568, donkey anti-chicken Alexa 647 (all 1:1,000, Invitrogen). Nuclear counterstaining was performed with 4', 6'-diamidino-2- phenylindole dihydrochloride hydrate at 0.25 μg μl⁻¹ (DAPI; Sigma). Specimens were mounted on microscope slides using a Prolong Antifade kit (Molecular Probes). Epifluorescence observation was performed on a confocal scanning laser microscope (LSM 710, Zeiss,) with LSM software (ZEN 2011).