Fla5100 reader

Wild-type and mutant strains were cultivated in supplemented liquid AMM for 16 h at 37 °C, after which the culture medium was made 1 M with respect to NaCl concentration or 10 mM with respect to CaCl₂ concentration where indicated. Mycelia were incubated for one hour and samples were collected at time 0 (absence of added NaCl or CaCl₂), 15, 30 and 60 min (presence of NaCl or CaCl₂ where indicated). Total RNA extraction and northern protocols followed (Garzia et al., 2009 (link)). Transcripts of sltA and sltB were detected using specific genomic probes amplified by PCR (oligonucleotides listed in Table S1). A 1573 bp probe (covering 56% of the ORF) was used for sltA and a 1452 bp probe (covering 35% of the ORF) was used for sltB. Transcript levels of the housekeeping glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) was used as internal control for normalization. A PhosphorImager imaging plate, BAS-IP-MS 2040, was used to detect radioactive mRNA bound probes with development using a FLA-5100 Reader (FujiFilm). Quantification of band intensities was performed using Multi-Gauge V3.0 software (Fujifilm).

RNA samples were extracted using TriReagent (Sigma-Aldrich Quimica, Madrid, Spain) and following the manufacturer's indications. A radioactive high prime DNA labeling kit (Sigma-Aldrich Quimica) was used for the generation of probes. The hybridization solution described by Church and Gilbert was used (1% BSA, 1 mM EDTA, 0.5 M NaPO₄, pH = 7.2 and 7% sodium dodecyl sulfate (SDS)) [27 (link)]. Hybridizations were performed overnight at 55 °C, and the filters were washed twice with a 2% saline-sodium citrate (SSC)/0.1% SDS solution at 55 °C and then with a 0.2% SSC/0.1% SDS solution, heated at the same temperature. Detection of sbtA, sbtB, enaA and pho89^An8956 transcription was performed by exposing the blots to a PhosphorImager screen (Molecular Dynamics, GE Healthcare Europe GmbH, Freiburg, Germany) and developing using a FLA-5100 Reader (Fujifilm Life Science, FujiFilm Europe GmbH, Barcelona, Spain).

After the nano-SPECT/CT studies, HCT-116 bearing subcutaneous tumor nude that had been injected ¹¹¹In-cetuximab after 48 hours. Whole body of nude mice were collected, frozen, and embedded in OCT compound (Tissue Tek, Sakura, Torrance, CA). Frozen sections were placed in contact with a BASMS 2040 imaging plate (Fujifilm, Japan) for thirty days. After complete exposure, the imaging plate was analyzed with an FLA-5100 reader (Fujifilm, Japan) and Multi Gauge V3.0 software (Fujifilm, Japan).
HCT-116/Luc bearing abdominal nude mice whole body of ¹¹¹In-cetuximab and ¹¹¹In-DTPA after injected 72 hours were collected, frozen, and embedded in OCT compound after the nano-SPECT/CT studies. The follow steps described as above paragraph.