Peloris automaton

Right ovaries were collected from female mice (used for biodistribution) on D15 and D180 and were preserved in 10% buffered formalin and then in 70% ethanol. Whole ovaries were processed in Peloris automaton (Leica) and then embedded in paraffin.
For each animal, 10 sections (3- to 5-μm thick) were manufactured and deposited on two Superfrost+ slides. One slide was treated with the AAV probe, which was designed on the BGH poly(A) region. The second slide was processed according to the same protocol without the AAV probe, which was the negative control. Each assay included positive controls (i.e., a slide of ovary labeled with a control mouse probe designed on the sequence of elongation factor 1 [EF1] and a slide of injected mouse liver labeled with the AAV probe).
Slides were scanned at ×20 magnification in bright-field conditions with a Nanozoomer scanner (Hamamatsu). The analysis consisted of the detection of a positive signal in the slide labeled with the AAV probe by comparison with the negative control slide. The RNA in situ hybridization analysis was performed by Histalim.

Samples were fixed in 4% formalin at room temperature for 24 to 36h, then moved into 70% ethanol after two washes with PBS to stop fixation and then kept in a refrigerator at 4°C.
The samples were processed according to a HIS 6h program in the Peloris automaton (Leica) for dehydration and then embedded in paraffin to obtain transversal sections through all abdominal wall layers. Paraffin wax blocks were cut into 3–5 mm thick slices and stained with hematoxylin-eosin. One histologist evaluated all tissues and was blinded to the origin of the meshes.

After removal, the organs were fixed in formalin for 24 h and transferred into 70% ethanol. To ensure a non-biased comparison, the same parts of the organs were analyzed by HISTALIM (Montpellier, France). The samples were processed on the Peloris automaton (Leica, Wetzlar, Germany) according to the 4 h program validated for mouse organs. The samples were embedded in paraffin wax according to HISTALIM procedures. For the liver and spleen samples, a section (3–5 μm thickness) was prepared and deposited preferentially on Superfrost + slide (to ensure tissue adhesion) to be stained according to a validated hematoxylin/eosin (H&E) protocol. All the slides were digitalized with the Nanozoomer scanner (Hamamatsu Photonics, Hamamatsu, Japan) in bright field, with the objective ×20, without Z stack.