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The percentage of double labelled cells versus BrdU+ cells was evaluated for PSA-NCAM, Pax2, Map5, Sox2.
The percentage of bipolar and intermediate or neuronal-like BrdU/PSA-NCAM double labelled cells was then compared between 5 and 15 days after BrdU injection.
Fluorescent images of single and double labelled tissue sections were recorded with a Zeiss LSM410 confocal microscope using a Zeiss 63x NA 1.25 oil-immersion objective.
In the present study, we found that fibers double labelled with PGP and mTOR did not penetrate the dermal-epidermal junction (Fig. 1A).
Accordingly, also the number of BrdU+/Sox2+ double labelled cells was very high between two weeks and two months after their genesis (Figure S2C).
For double labelled sections, images were collected using sequential acquisition between stacks.
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Qualitatively, our immunofluorescence images are very similar compared to semi-thin cyrosections from mouse tibialis anterior muscle double-labelled for dystrophin and α-actinin (Fig. 3 in [60]).
To gain more insight into the intracellular pool, we double-labelled cells with different cytoplasmic organelle markers.
To study the effects on phagosome processing (maturation) of loss of CLN3, we double-labelled retinal sections taken 2 h after light onset.
Since progenitor cells committed to interneuron production express the transcription factor Dlx2 (Flames and Marin, 2005), we double-labelled sections with NeuN and Dlx2.
Within these three zones, immunopositive cells (excluding any vascular labelling) and double-labelled cells were counted at a high magnification (×40) by one researcher (J.G).
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