(a, b) EM images of the spinal cord dorsal horn of the same ThT2A-CreER animal transduced with AAV9-DIO-Matrix-dAPEX2 and treated with tamoxifen at P14–21 to label C-LTMRs prepared with the rOTO protocol (a) or the reduced osmium protocol (b). Strong spurious staining was seen in the IMS in the samples prepared with the rOTO protocol. This spurious staining was not seen in unlabeled mitochondria. Reduced osmium staining preserved the DAB staining while providing sufficient counterstaining contrast. n = 2 animals and experiments. (c) EM images showing a sample prepared with the rOTO protocol with double labeling of cortical layer 5 pyramidal neurons (ER) using Tg(Rbp4-Cre)KL100 and AAV1-DIO-ER-dAPEX2 (red asterisks), and fast-spiking GABAergic interneurons (mitochondrial matrix) using PvalbT2A-FlpO and AAV1-FDIO-Matrix-dAPEX2 (green asterisks), equivalent to the experiment in Fig. 3a. Arrowhead: symmetric perisomatic synapse made by fast-spiking interneurons onto layer 5 pyramidal neurons. Note while spurious staining was present in the IMS, the mitochondrial matrix staining could still be easily distinguished from the ER staining. n = 2 animals and experiments. Scale bars: a, b: 0.2 μm, c: 0.5 μm.