Nam and Capecchi, 2020

Materials from and additional supplementary data in support of the Nam and Capecchi, 2020 paper:

(1) Some of the high resolution images that were quantitated for graphs

(2) Data that were mentioned but not included in the paper ("data not shown")

(3) Control experiments with additional discussion of the technical details

etc.

Line 1c2 Lrig1-T2A-iCreERT2 mouse was utilized except for experiments in Figures 2H-2J, 2W-2X.

✻

Mice that were induced with a single injection of low dose tamoxifen at 3 months of age showed clusters of proliferating cells at 1 year after the induction (Figure 3I in Nam and Capecchi, 2020, also see here). The combination of the Lrig1-T2A-iCreERT2 allele and Rosa26-Ai14 allele did not leak significantly without the tamoxifen induction (see here). Thus, we can conclude that the cells revealed by the RFP label at early time points (see here and here) include the long-term neurogenic stem cell activity. As discussed in the paper, the RFP+ cells included cells with very distinctive morphologies, what we termed the α and β morphologies. By additional experiments, these cells were demonstrated to be the only cells among the RFP-labeled cells capable of quiescence exit, cell cycle entry, and proliferation to give rise to differentiated progeny cells. Thus, the Lrig1-T2A-iCreERT2 reporter allele reveals the cells with the distinctive morphologies as long-term neurogenic stem cells. These cells were observed throughout the lateral ventricular wall and on average located deeper in the ventricular wall.

See here for additional comments.

❋

By the way, all immunohistology on whole mounts or cryosections were done on brains fixed with PLP fixative with 2% formaldehyde. In the very early days of this postdoctoral training project, a search through the internet for fixatives yielded the PLP fixative as well as other fixatives such as the zinc fixative. The PLP fixative with 2% formaldehyde worked great from the first time. More or less every antibody I tried with it worked well, even the mouse anti-ASCL1 (MASH1) antibody. However, the same fixative with 4% formaldehyde, in contrast to the 2% formaldehyde, was a different story. Many of the same antibodies didn't work on the brains fixed with the PLP fixative with 4% formaldehyde (see this file, clicking the link will download a file). Thus, the lower formaldehyde concentration was the detail that made the difference.

Furthermore, in early immunostainings done with a protocol adapted from the MSKCC Molecular Cytology Core Facility protocol, 2% BSA block was sufficient to also block reactive aldehydes after formaldehyde fixation. However, at some point, I noticed that the normal IgG antibody and F(ab) antibody fragment weren't binding so well in the 2% BSA buffer. So, in later immunostainings, the BSA was reduced to 0.5% to increase their binding. Then, the antibodies bound better, but the 0.5% BSA was insufficient for block of the aldehydes, so an additional block with 0.3 M glycine in PBS pH 7.4 was added before the permeabilization step.

By the way, after the post-fix and the glycine block, the brains can be stored refrigerated in the glycine in PBS buffer for long-term. If handled carefully to maintain sterility, the brains were stable for more than a year. When ready to do the staining, transfer the brains to 0.5% TX-100 in PBS for permeabilization, and so on.

See here for fixative and buffer recipes (clicking the link will download a file).