DOI: 10.6084/m9.figshare.30574283.v4
title: Lateral ventricular wall whole mount immunofluorescence from brains of Ara-C-infused <i>Lrig1</i><sup><em>T2A-iCreERT2/+</em></sup><i>; Rosa26</i><sup><em>Ai14/+</em></sup> mice

description: Dataset from the Ara-C infusion experiment presented in Nam and Capecchi, 2020.The ZIP files can be opened directly with Fiji.✶Ara-C is a classical nucleoside chemotherapeutic that kills dividing cells.Ara-C was slowly infused over six days into one of the two lateral ventricles using a cannula and an osmotic pump, in contrast to infusion into the cortex as in Doetsch et al., 1999 and Doetsch et al., 1999.Recall that Ara-C doesn't directly induce cell cycle entry of quiescent stem cells. It does so indirectly by killing nearby proliferating cells, and their absence induces the quiescent stem cells into cell cycle. As such, the cell cycle entry does not necessarily happen exactly when the infusion is stopped, but rather when a sufficient number of cells is killed by the Ara-C. Indeed, in my implementation of this paradigm, it would seem that the cell cycle entry of the quiescent stem cells had started before the cessation of the Ara-C infusion because at that time point, the RFP+ quiescent stem cells were already KI-67+, i.e, activated. A key difference indicating that the Ara-C infusion (indirectly) caused the cell cycle entry is the greatly increased number of these RFP+ KI-67+ activated stem cells in the infused mice compared to uninfused mice.There were some neuroblasts that had not been killed by the Ara-C. Subsequently, I noticed there are KI-67+ DCX+ and KI-67- DCX+ neuroblasts (in Nam and Capecchi, 2023). Perhaps the KI-67- DCX+ neuroblasts are post-mitotic and cannot be killed by the Ara-C, but they will migrate rostrally and out of the ventricular wall during the chase after the infusion.One of the mice in the first try was not infused correctly because the tubing that connects the osmotic pump and the infusion cannula came loose after surgery. This could be considered an incidental sham surgery control. Note the presence of RFP+ proliferating clusters and RFP+ neuroblasts in the uninfused mouse. They are absent in the Ara-C-infused mice at early time points because they were killed by the Ara-C. That and the dramatic reduction of DCX+ neuroblast number indicate a successful Ara-C infusion.One of the two ventricular walls in one of the mice in the first try was damaged. At the time, I assumed I damaged the tissue during dissection and didn't think much more about it, but the damage could be due to the Ara-C infusion (as recently reported in Thanou et al., 2025). Perhaps in this mouse the infusion cannula was placed too close to the ventricular wall, and the high concentration Ara-C flowing out of the cannula was too toxic to the nearby cells and damaged the tissue. In the first try, I analyzed both ventricular walls, and the damage was observed in only one of the two successful infusions. In the second try, I unfortunately did not analyze both ventricular walls. I randomly chose one of the two, then analyzed only one. Nevertheless, none in this set were damaged. In the third try, I analyzed both ventricular walls from one mouse, and neither were damaged. So, I will not know for sure from this dataset, but the damage doesn't seem to happen in every mouse using this particular setup.These were done very early on in my post-doctoral training, so the immunostaining quality is not as good as the ones done later.