Bovine serum albumin (bsa)

Immunocytochemistry procedure was performed based on protocols described elsewhere [22 (link), 23 (link)]. Briefly, for cells harvest, we washed cultures with PBS and fixed it with 4% PFA for 20 min. Cells were permeabilized with 1% Triton™ X-100 (Sigma) and blocked using 3% Bovine Serum Albumin (Nzytech) in PBS. Incubation with primary antibodies was done overnight at 4 °C. After PBS washes for antibodies removal, cells were incubated in secondary antibodies for 2h at room temperature (RT). Finally, cells were washed with PBS and coverslips were mounted on microscope slides using Fluoromount-G™ medium with DAPI (4′,6′-Diamidino-2′-phenylindole) (ThermoFisher Scientific). Images were acquired with 40x objective in a Andor benchtop spinning disk confocal instrument (Oxford Instruments, UK) and with a 63x and 100x objectives in a Zeiss LSM710 confocal microscope.

Protein concentration was determined by the Bradford Method [50 (link)], using the Bio-Rad protein assay dye reagent concentrate (BioRad, Hercules, CA, USA) in cellular lysates and retinal tissue. Bovine serum albumin (NZYTech, Lisboa, Portugal or Thermo Fisher Scientific, Waltham, MA, USA) was used as standard (1 mg/mL; 0.5 mg/mL; 0.25 mg/mL; 0.125 mg/mL; and 0.0625 mg/mL) prepared in miliQ water. Samples were also diluted in miliQ water in 1:5 or 1:10 ratios for cell lysates and retinas, respectively. The cell and standard samples were loaded into a 96-well plate, and the dye reagent added to each well. The absorbance was read in a Biotrak II Plate reader (Amersham Biosciences, Amersham, UK) at 595 nm and concentration of protein was determined by linear regression.

Retinas were dissected as previously described (del Toro et al., 2010 (link)). In brief, eyeballs were fixed for 18 min in 4% paraformaldehyde at room temperature. After dissection, retinas were blocked for 1 hr in blocking buffer (TNBT) or Claudio’s Blocking Buffer (CBB) for retinas with Golgi stained. CBB consists of 1% FBS (ThermoFisher Scientific), 3% BSA (Nzytech), 0.5% Triton X100 (Sigma), 0.01% Sodium deoxycholate (Sigma), 0,02% Sodium Azide (Sigma) in PBS pH = 7.4 for 2 hr in a rocking platform) for retinas stained for golgi. Thereafter, retinas were incubated overnight in primary antibody in blocking buffer. After extensive washing, retinas were incubated in the corresponding secondary antibody for 2 hr at room temperature. For confocal microscopy, retinas were mounted on glass slides, and for LSFM, retinas were mounted in 2% low-melting agarose. Agarose was melted at >65 °C, and then maintained at 42 °C before adding the tissue. To minimise curling of the retina, apply 1–2 drops of low melting agarose on retina and start uncurling the retina before the gel is solidified. It can then be transferred to the cylinder for imaging.

Zymogram assay for detection of bacteriolytic activity of the putative lysins were performed as previously described (2011) [70 (link)], with adjustments considering the use of Gram-negatives that have thinner layer of peptidoglycan. For cell preparation, M. luteus and P. aeruginosa were grown in LB Broth, at 37 °C, until stationary phase. Cells were collected, suspended in water, and then autoclaved. The autoclaved cells were resuspended in water to a final concentration 2% (dry weight). For the Gram-positive M. luteus, 0.2% cell preparation, and for the Gram-negative bacteria P. aeruginosa, 0.4% cell preparation were incorporated on the 12% polyacrylamide gel. After electrophoresis, the zymogram gels were incubated in a renaturation buffer (25 mM Tris-HCl pH 7.5 and 1% Triton X-100) overnight at 37 °C, with mild agitation. The gels were stained for 90 min in zymogram staining solution (0.5% Methylene Blue and 0.01% KOH) and distained in distilled water. Lysozyme of egg white (1 µg) (Sigma-Aldrich, St. Louis, MO, USA) and BSA (5 µg) (NZYTech, Lisbon, Portugal) were used as positive and negative control, respectively. For migration control, a regular SDS-PAGE was performed in parallel and stained with BlueSafe (NZYTech, Lisbon, Portugal).

Forty-eight hours after transfection, the medium was removed and the cells were washed with PBS and fixed with 4% PFA for 30 min at RT. Cell permeabilization was performed with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at RT, followed by blocking in 3% BSA (Nzytech, Lisbon, Portugal) in PBS 1× for 1 h. Afterward, cells were incubated with primary antibodies: mouse anti-ASY (1:1,000, BD Transduction Laboratories, Franklin Lakes, NJ, USA) and rabbit anti-CDNF (1:1000, Sigma-Aldrich, St. Louis, MO, USA) overnight and secondary antibody Alexa Fluor 488 donkey anti-mouse IgG (Life Technologies-Invitrogen, Carlsbad, CA, USA) for 2 h at RT. Finally, cells were stained with DAPI (Carl Roth, Karlsruhe, Germany) (1:5,000 in PBS 1×) for 10 min, and the coverslips mounted on SuperFrost Microscope Slides treated with Mowiol (Calbiochem, San Diego, CA, USA), dried and stored at RT until further visualization and analysis. Images were acquired using an epifluorescence microscope (Leica DMI 6000B microscope, Leica, Wetzlar, Germany). For each condition, 20 images were taken with the same exposition time for each channel. Normalized image analysis was performed using Fiji⁵⁷ (link) open-source software using the region of interest (ROI) plugin and calculating the mean fluorescence intensity of each ROI.

Western blot analysis was performed with spent medium and cell extract samples collected during cell culture growth. Medium samples were 10-fold concentrated using centrifugal filtration units with 10 kDa cut-off Amicon Ultra-15 (Millipore, USA). Cells were ground using liquid nitrogen and a mortar. Extraction buffer (100 mM ascorbic acid, 500 mM NaCl, 5 mM β-mercaptoethanol, pH 8.0) was added to ground cells in 1:1 ratio (g of cells: mL of extraction buffer). Samples were resolved in a 12.5% SDS-PAGE gel and proteins were transferred to a nitrocellulose membrane (0.2 µm, Amersham, UK). The membrane was blocked with 5% skimmed milk powder (Nestlé, Switzerland) and 3% BSA (NZYTech, Portugal) in PBS-T, for 1 hour at room temperature with gentle shaking, followed by incubation with 1:500 anti-L-PGDS (Ab 61866, Abcam, UK) in PBS-T, overnight at 4 °C. Then, the membrane was incubated with 1:8000 HRP conjugated anti-rabbit (A0545, Sigma-Aldrich, USA) in PBS-T, for 2 hours at room temperature with gentle shaking. Signal was detected by chemiluminescence using Amersham ECL Prime reagent (GE Healthcare Life Sciences, UK) in ChemiDoc™ XRS + System (Bio-Rad, USA). Three biological replicates were analyzed and western blots repeated three times.