Extended Data Fig. 1 |. Immunohistochemical identification of interneuron subtypes and separation of CA2 and CA3 interneurons with anti-STEP immunohistochemistry.

(a) Schematic of the experimental pipeline used to determine the molecular identity of imaged cells. Multiple rounds of immunohistochemistry were performed on fixed, horizontal slices that were registered to high-resolution in vivo Z-stacks. (b) Example in vivo 2p-AOD image (left) and confocal image (right) of the registered FOV. White arrows indicate the registered cells. Calbindin immunohistochemistry was used to label the mossy fibers of stratum lucidum of CA3/CA2. This procedure was repeated in n = 22 imaged mice. Scale bars on the left and right images represent 50 and 100 μm, respectively. (c) Example labeling strategy used to determine the subtype and region identity of imaged cells. Immunohistochemical labels were not removed between the different rounds (see Methods). (d) Example immunohistochemical labeling and combinatorial expression patterns of the 5 markers (PV, SOM, SATB1, CCK, CB) used to separate imaged cells into subtypes. This procedure was repeated in n = 22 imaged mice. All images are approximately 60 × 60 μm. (e) CA2 interneurons were identified by their proximity to STEP-expressing CA2 pyramidal cells (top row). In comparison, CA3 interneurons occupied slices where Calbindin-positive mossy fibers were present but where the majority of pyramidal cells were not STEP-expressing (bottom row). This procedure was repeated in n = 22 imaged mice. Scale bars on all four images represent approximately 100 μm.