# Novel Phenotypic Elements of Type IV Collagenopathy Revealed by the _Drosophila_ Model
## 5 Materials and Methods  
Wild-type _Oregon_ flies and _col4a1_ mutant lines were maintained at 20 \({}^{\circ}\)C and 29 \({}^{\circ}\)C on yeast-commaneal-sucrose-agar food, consisting of nipagin to prevent fungal infection. The mutant stocks were kept heterozygous over the recombination--preventing _CyRoi_ balancer chromosome. Malpighian tubules, common oviducts and the gut were dissected under carbon dioxide anesthesia from adults that were grown at either the permissive or the restrictive temperature for 14 days. Dissected organs were fixed in a previously prepared 4% paraformaldehyde dissolved in phosphate buffered saline (PBS) solution for 10 min, washed three times in PBS, permeabilized for 5 min in 0.1% Triton-X dissolved in PBS solution and washed three times in PBS. Blocking was achieved in a 5% bovine serum albumin dissolved in PBS solution for 1 h, and washed three times in PBS.  
Complementation tests were performed by ethyl-methanesulfonate-induced point mutants, P-element insertional mutants and X-ray-generated deletion (null) alleles [24]. The mutation sites in eight temperature-sensitive lines were sequenced. All mutations were G to A transitions and replaced glycine with glutamate, aspartate and serine. The _a-30_ allele carried the p.G233E amino acid substitution; the _b-9_ carried the p.G467E substitution. The isoallelic lines _DTS-L2_ and _DTS-L3_ carried the p.G552D1 and p.G552D2 substitutions, respectively. The second isoallelic pair _DTS-L4_ and _DTS-L5_ carried the p.G1205E1 and p.G1205E2 substitutions, respectively, and the _DTS-L10_ carried the p.G1043S substitution, whereas the _b-17_ carried the p.G193E substitution. Importantly, the lesions covered the collagenous domain of the COL4A1 protein [29].  
Nuclei in the dissected organs were counter-stained by 1 ug/mL 4\({}^{\prime}\),6-diamino-2-phenylindol (DAPI) in 20 \(\upmu\)L PBS for 12 min in the dark. F-actin was stained by 1 unit Texas RedTM-X Phalloidin (ThermoFisher) in 20 \(\upmu\)L PBS for 20 min. Next, 1 \(\upmu\)L mouse monoclonal anti-3-Nitrothyrosine, anti-integrin alpha PS and anti-kettin antibodies (Abcam) were used in 20 \(\upmu\)L PBS for 1 h. Mouse anti-COL4A1 antibodies were raised by Creative Laboratory Ltd., Szeged, Hungary. Primary mouse antibodies were visualized by 1 \(\upmu\)L F(ab') 2-Goat Anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody conjugated with Alexa Fluor 488 (ThermoFisher) in 20 \(\upmu\)L PBS for 1 h and 1 \(\upmu\)L Goat Anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody, Alexa Fluor 350, in 20 \(\upmu\)L PBS for 1 h. Mitochondria were visualized by the mitochondally targeted enhanced yellow fluorescent protein (mito-GFP) following appropriate crosses.  
Photomicrographs were generated by confocal laser scanning fluorescence microscopy (Olympus Life Science Europa GmbH, Hamburg, Germany). The microscope configuration is described as follows. Objective lens: UPLSAPO 60\(\times\) (oil, NA: 1.35); sampling speed: 8 us/pixel; line averaging: 2\(\times\); scanning  
Figure 4: Loss of sarcomere structure in visceral muscle fibers. Regular sarcomere structure in the wild-type control at both temperatures (**A1**, **A2**, **A3** and **A4**, **A5**, **A6**) and loss of sarcomeres in the mutant that deposits actin in an irregular fashion (**B1**, **B2**, **B3** and **B4**, **B5**, **B6**). Bars, lower left: 10 micrometers. Experiments were carried out as described [24; 25; 26; 28; 29].  
mode: sequential unidirectional; excitation: 405 nm (DAPI), 543 nm (Texas Red) and 488 nm (Alexa Fluor 488); laser transmissivity: 7% was used for DAPI, 42% for Alexa Fluor 488 and 52% for Texas Red.