Metamorph offline

To determine the load of CAB101 and CAB102.1 per nigral dopamine neuron we performed a semiquantitative analysis scoring the overlap of CAB101 and CAB102.1 with neuromelanin-containing neurons. A blinded investigator scored the level of overlap of either CAB101 or CAB102.1 with neuromelanin on a score from 0 (no overlap of cells) to 10 (complete overlap). Three sections were analyzed per case and the averaged used to generate the overall score per case. For the integrated intensity analysis, the same sections were imaged as described above and were analyzed using Metamorph software (Metamorph Offline v.7.8.0, Molecular Devices). Image analysis was conducted using the “Count Nuclei” algorithm and the integrated intensity measurements were logged.

For colony formation assays, H1299 cells were transfected with the indicated plasmids and grown in appropriate selection medium (G418) for between 10 and 14 days. Surviving colonies were stained with crystal violet solution (Merck) as described previously (33 (link)). Quantifications were done using MetaMorph Offline (Version 7.8.0; Molecular Devices, LLC), which quantifies live cells by percent colony area.

The differentiated 3T3-L1 adipocytes were fixed with 10% formalin, washed with 70% ethanol and phosphate-buffered saline (PBS), and finally stained with ORO solution. Stained lipid droplets were visualized using an Olympus CKX41 inverted microscopy (Olympus, Tokyo, Japan). We calculated the number of ORO stained lipid droplets for each group using Metamorph offline (Molecular Devices Co., Sunnyvale, CA). Cells were exposed to OYSGS for 8 days during adipocyte differentiation.

Merged multichannel 40× light microscopy images were analyzed using MetaMorph Offline Version 7.7.0.0 (Molecular Devices, Inc., San Jose, CA, United States). A threshold is set for each channel followed by the generation of a mask for all channels in three areas of size 25 pixels long (representing 2 μm in the sample) and 4 pixels wide, per image. These area masks were then overlaid in the arithmetic tool and divided to generate a third mask containing only the population of fluorescence signals in the mask that do colocalize. The amount of bassoon colocalized is represented as a percentage of the bassoon-colocalized population divided by the total bassoon population.

The femur samples were harvested in neutral buffered formalin (10%), incubated in a rapid decalcifier solution, trimmed, and embedded in paraffin. Subsequently, 4 mm thick sections were stained with (1) hematoxylin/eosin (H&E), (2) Masson’s trichrome, and (3) cathepsin K (1:100, ab19027, Cambridge, MA, Abcam). Primary antibodies against cathepsin K (1:100, ab19027, Abcam, USA) were used for immunofluorescence staining. Samples were subsequently incubated with a secondary antibody, namely Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, A21206, Invitrogen, Carlsbad, CA, USA), for 60 min at 25 °C.
The signal of osteoclasts occurs around the trabecular bone area. We used MetaMorph Offline (v7.8.13.0, Molecular Devices, Sunnyvale, CA, USA) to quantify the cathepsin K signal in the trabecular bone region as follows: (1) We used the software to quantify the black area representing the trabecular bone. (2) The green fluorescent signal represented cathepsin-K-positive osteoclasts; thus, we quantified the green fluorescent signal. (3) The value obtained by dividing green fluorescence signals by the trabecular bone area represented the intensity of cathepsin-K-positive osteoclasts in the trabecular bone. 4) Each group had five or six mice, and three photos were taken in different areas in each femur for quantification.