Figure 2. Peroxidase constructs targeted to different subcellular compartments for multiplexed EM labeling.
(a) EM images showing localization of dAPEX2. Asterisks: labeled neurons. Staining in the cytoplasm is often not uniform and can appear granular. (Left) Soma of a cortical layer 5 neuron labeled using Tg(Rbp4-Cre)KL100. Red arrow: labeled cytoplasm. Blue arrow: unlabeled cytoplasm. Note that membrane-limited organelles, such as ER (green arrow), mitochondria, and Golgi apparatus, can usually be distinguished in stained cells. (Middle) Dendrites of cortical layer 5 neurons labeled using Tg(Rbp4-Cre)KL100. (Right) Axon of a primary sensory neuron in the spinal cord dorsal horn after AAV9 systemic transduction. Arrowheads: synapses made by the labeled neuron. n = 2 animals and experiments for each condition.
(b) EM images showing localization of ER-dAPEX2. Asterisks: labeled neurons. (Left) Soma of a cortical layer 5 neuron labeled using Tg(Rbp4-Cre)KL100. Red arrow: labeled ER. Blue arrow: unlabeled Golgi apparatus. Note that nuclear envelope is labeled as expected and nuclear pores (green arrows) are clearly visible, unobscured by the reaction product. (Middle) Inhibitory interneurons in the dorsal horn labeled using Slc32a1IRES-Cre. Arrowhead: a synapse received by an inhibitory interneuron. (Right) Inhibitory interneurons in the spinal cord dorsal horn labeled using Slc32a1IRES-Cre. Arrowhead: a synapse made by an inhibitory interneuron. Note that identification of small ER profiles can be difficult and only clearly identified profiles are marked. n = 2 animals and experiments for each condition.
(c) EM images showing localization of IMS-dAPEX2. Asterisks: labeled neurons. (Left) Soma of a cortical layer 5 neuron labeled using Tg(Rbp4-Cre)KL100. Red arrow: labeled mitochondrion. Blue arrow: unlabeled mitochondrion. Preservation of the full extent of IMS staining is not always achieved, potentially due to difficulty in sectioning dense heavy metal labeling, however this usually does not hinder identification of stained mitochondria. (Middle) Dendrite of cortical layer 5 neuron labeled using Tg(Rbp4-Cre)KL100. (Right) Axon in the spinal cord dorsal horn after AAV9 systemic transduction. Arrowhead: synapse made by the labeled neuron. n = 2 animals and experiments for each condition.
(d) EM images showing localization of SV-HRP. Asterisks: labeled neurons. Not every vesicle in transduced cells is stained. (Left) Corticocortical axon of a cortical layer 5 neuron labeled using Tg(Rbp4-Cre)KL100. Red arrow: labeled vesicle. Blue arrow: unlabeled vesicle. Arrowhead: synapse made by the labeled neuron. (Middle) Corticospinal axon in the spinal cord dorsal horn of a cortical layer 5 neuron labeled using Tg(Rbp4-Cre)KL100. Arrowhead: synapse made by the labeled neuron. (Right) Axon in the spinal cord dorsal horn after AAV9 systemic transduction. n = 2 animals and experiments for each condition.
Scale bars: 0.5 μm.