49]) or B1 (detecting free capsid proteins of all AAV serotypes used here except AAV4) ,
GeneTex) was diluted 1:500 in the hybridoma supernatants Cells were then washed twice for 10 min each with IF blocking buffer and then incubated for 1 h at 37°C with secondary antibodies diluted 1:700 in blocking buffer. To quantify B1 and A20/ADK stainings, an anti-mouse AF488-labeled secondary antibody (Life Technologies) was used. The anti-HA antibody was detected with an anti-rabbit AF647- labeled secondary antibody (Life Technologies) To stain nuclei, Hoechst stain (Molecular Probes, Thermo Fisher Scientific) diluted 1:3,000 was added to all samples. Next, cells were washed with 1 PBS and stored at 4°C in the dark in 200 l 1 PBS per well, HeLa cells were treated the same way, except that cells were analyzed at 42 h posttransfection and stained with the anti-HA antibody (and Hoechst stain, 29110. ,
75 l H 2 O. Reactions were run in a StepOnePlus real-time PCR system (Thermo Fisher Scientific) using the following program: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min, and a final melting step from 60°C to 95°C in increments of 0.3°C. Samples were measured in duplicates and normalized to a GAPDH housekeeper (primer pair 1117 For the final analysis, 1118. ,
Thermo Fisher Scientific, and produced by immunization of rabbits with peptide GKDSST TTGDSDPRDSTS (AAP2 residues 121 to 138) (17) Affinity purification yielded the final polyclonal anti-AAP2 antibody (preserved in 1% BSA), which was used for Western blot analyses of insect cell samples and for immunostainings. Dot blot analysis. Crude cell lysates containing AAV particles were produced as stated above. A nitrocellulose membrane (Thermo Fisher Scientific) was cut to size, soaked in PBS for 10 min, and assembled into a Bio-Dot microfiltration apparatus (Bio-Rad) according to the manufacturer's protocol. Next, 15 l of the indicated crude lysates was loaded. To promote capsid disassembly for detection of free, unassembled viral capsid proteins, 100 l of each sample was heated to 95°C for 5 min, and 15 l REFERENCES 1 Adenovirus-associated defective virus particles, The anti-AAP2 antibody was ordered from the company Perbio, pp.754-756, 1965. ,
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