Neurons were washed with PBS/30 mM glycine pH 2.5 to remove the remaining surface label, fixed, and immunolabeled using antibodies against Tmub1/HOPS or VAMP2.
Coverslips were washed to remove the excess of labelling, fixed with 4% PFA for 10 min, rewashed with PBS, and then mounted.
Cells were labelled, fixed with 1% paraformaldehyde and stained with anti-IL-12-PE in 0.2% saponin/PBS at 4°C overnight.
To examine whether the two subjects showed differences in dividing cells within the SVZ, we labeled fixed tissue samples with Ki67 to mark cycling cells.
Healthy volunteers (six men and six nonpregnant women) were enrolled in an open-label, fixed sequence crossover study conducted at the UNC CTRC.
To test this, we transiently transfected cytochrome c GFP expressing HeLa cells with N320 GRASP65–myc or ΔN320 GRand65–myc, and labelled fixed cells with anti-myc antibodies.
Discs were dissected and incubated in 10 μM EdU in Ringer's for 20 min or 2 h and, following EdU labeling, fixed and immunostained as described in Tortoriello et al. Afterwards, they were incubated in 1x Click-iT reaction cocktail for 30 min, washed thoroughly in PBS and mounted.
For BrdU labelling, fixed neurons were incubated with 2 N HCl for 30 min at room temperature to denature DNA, followed by 0.1 M sodium tetraborate 5 min and then washed several times in PBS, before proceeding with the primary antibody incubation.
The patient labels were fixed as positive class labels.
But this is not possible in the standard multi-label formulation because it is transductive the labels are fixed during the training phase, and predictions on new labels are not possible.
