Imagemaster vds

Retina and sclera tissues were homogenized, respectively, and 5 μg of homogenate was loaded for SDS-PAGE. Proteins were separated on a 10% resolving gel and transferred to poly cellulose acetate membrane (Millipore).Then these membranes were blocked with 5% nonfat dry milk in 0.1% Tween 20 (TBS-T; 2 mmol/L Tris-HCl, 50 mmol/L NaCl, pH 7.4) for 2 hours at a room temperature. Afterwards, these membranes were then cultured overnight with a primary antibody against TGF-β, HAS, or GAPDH at a 1 : 1000 dilution at the temperature of 4°C in the blocked buffer. Furthermore, these membranes were washed with 0.1% Tween 20 and then treated with goat anti-rabbit IgG conjugated to alkaline phosphatase (1 : 5000) for 1 hour at the temperature of 37°C. Stripping filters and reprobing for GAPDH were normalized. The controls of nonspecific binding were determined by the absence of primary antibodies. A film scanner (Image Master VDS; Amersham Biosciences Inc., Piscataway, NJ) was used to scan the films.

Total community DNA was used as the template for amplification of the partial 16S rRNA gene fragment using Taq DNA polymerase (Bioline, Lückenwalde, Germany) with primer F968 with a GC clamp attached to the 5′ end and universal bacterial primer R1401.1b. For ITS1 amplification, primers EF4/ITS4 were used; this PCR was followed by a second amplification with primers ITS1f-GCITS2. Primer sequences, the reactions mixtures, and cycling conditions have been described (Pereira e Silva et al., 2012 (link)). The DGGE was performed in 6 % (w/v) polyacrylamide gels with 45–65 % and 20–50 % denaturant gradients for bacterial and fungal communities, respectively (100 % denaturant is defined as 7.0 M urea with 40 % deionized formamide). Electrophoresis was carried out at 100 V and 75 mA, for 16 h at 60 °C. The gels were subsequently stained for 40 min in 0.5 % TAE buffer with SYBR gold (final concentration 0.5 μg/L) (Invitrogen, Breda, the Netherlands) (Fig. S1 in the Supplementary Material). Gel images were digitized using Imagemaster VDS (Amersham Biosciences, Buckinghamshire, UK). The DGGE patterns were then transformed to a band-matching table using GelCompar II software (Applied Maths, Sint Martens Latem, Belgium).

The DGGE analysis was performed to estimate differences in the structure of bacterial communities across populations of D. melanogaster and to determine the sampling effort needed to fully characterize their community composition. 16S rRNA bacterial genes were PCR-amplified using the primer set F968 with a GC-clamp attached to 5’ and R1401 (S1 Table), as described above. The obtained amplicons were further used for the DGGE analysis. The DGGE were visualized with Imagemaster VDS (Amersham Biosciences, Buckinghamshire, United Kingdom) and further analysed with GelCompar software (Applied Maths, Sint-Martens Latem, Belgium). The observed low bacterial diversity, as evidenced by the low number of bands on DGGE gels (S1 Fig), justifies our choice of sequencing method (Sanger sequencing) and was used to determine sampling size.