Computational protocol: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

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Protocol publication

[…] Retinas were dissected in phosphate-buffered saline (PBS; pH 7.4), fixed in 4% paraformaldehyde in PBS for 30 min and blocked in PBS plus 0.3% Triton X-100 (PBST) with 5% goat serum for 30 min. Retinas were incubated with primary antibodies against Rh1 (mouse monoclonal 4C5, 1:200, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:200, Invitrogen), RFP (rat, 1:200, ChromoTek, Planegg, Germany) or PDH (rabbit, 1:200; from Dr. Craig Montell; ) in PBS plus 0.1% Triton X-100 (PBST) with 5% goat serum at 4°C overnight. Secondary antibodies against mouse, rabbit or rat IgG labeled with Alexa Fluor 488, Alexa Fluor 568 or Alexa Fluor 647 were used (1:500, Invitrogen). Samples were examined and images were recorded by using a Nikon A1-R confocal microscope and a Nikon eclipse Ni-U microscope (Nikon, Tokyo, Japan). Acquired images were processed by using Photoshop CS4 software and ImageJ. White-eyed flies were used in all immunohistochemistry experiments to avoid autofluorescence caused by eye pigmentation. No significant fluorescence was detected in any channels when the tissues were stained with secondary antibody only. The percentage of colocalization was calculated by using in the software Imaris X64 7.4.2 (Bitplane, Zurich, Switzerland). Briefly, protein-A-positive and protein-B-positive vesicles were marked by red and green dots, respectively. The total numbers of red, green and double-labeled dots were counted, and the corresponding percentages were calculated. [...] Total RNA was purified by using Trizol reagent. The RNA integrity was checked by using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with a minimum RNA integrity number of 8. The mRNA was enriched by using oligo magnetic beads (Invitrogen) and fragmented to ∼150–250 bp. cDNAs were synthesized by using random hexamer primers and purified by using a MinElute PCR purification kit (Qiagen, Valencia, CA). The 42-cycle single-end sequencing was performed by using an Illumina Genome Analyzer IIx. CASAVA pipeline v1.8 was then used for sequence extraction and filtering. RNA-Seq reads were mapped to the fly genome by using the Tophat (v2.0.8b) software and the Ensembl genome annotation dataset (Drosophila_melanogaster.BDGP5.71.gtf). Then, the gene expression level fragments per kilobase of exon per million fragments mapped (FPKM) was estimated by using Cufflinks (v2.1.1) software. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster
Diseases Retinal Degeneration