Journal: STAR Protocols

Article Title: Protocol for whole-cell patch-clamp recording and post hoc identification of hippocampal CA2 pyramidal neurons in adult mouse brain slices

doi: 10.1016/j.xpro.2026.104470

Figure Lengend Snippet: Patch-clamp recording system (A) Overview of the preparation table showing the slicer at the left, two PP beakers on ice containing sucrose solution supplied with 95% O2 and 5% CO2. At the foreground a 80 mm in diameter crystallizing dish containing sucrose solution oxygenated using 95% O2 – 5% CO2 is to receive the brain after resection. Large scissors are to remove the head and small scissors to open the skull with a caudal to nasal cut. (B) Schematic representation of the sequential cuts to prepare hemispheres. View of a mouse brain from the top. The cerebral cortex is in grey, the cerebellum in yellow and the hippocampal region in green. A first cut (1) using a scalpel is to remove the cerebellum. A second sagittal cut (2) is to separate both hemispheres. A third (3) and a fourth (4) cut are to remove a small piece of tissue at the lateral side of the hemispheres. The lateral side of the hemisphere is glued on the specimen disk. Three-dimensional structure of adult mouse from Brain Explorer 2 (version 2.3.5 Built 2393, Allen institute, https://brain-map.org ). (C) View of the medial side of the two hemispheres in a Petri dish after sagittal cut of the whole brain. The base of the Petri dish is filled with hardened agarose. Brain tissue is lying on the agarose layer and surrounded by liquid-solid sludgy sucrose solution. Inset shows an enlarged view of the hemispheres. (D) View of the top of a mouse brain hemisphere. The brain is in gray and the hippocampus (HPC) in green. Parasagittal slices are cut starting from the medial side of the hemisphere towards the lateral side. Red dashed lines represent consecutive cuts to produce 300 μm thick slices containing the dorsal hippocampus (dorsal HPC). Three-dimensional structure of adult mouse from Brain Explorer 2 (version 2.3.5 Built 2393, Allen institute, https://brain-map.org ). (E) Cutting of brain slices in the parasagittal plane using the vibratome. View of the buffer tray containing sucrose solution with a liquid phase close to the hemispheres and a liquid-solid sludgy phase at the border of the buffer tray. (F) Magnified view of the buffer tray. Hemispheres are glued on a specimen disk. The specimen disk contains a magnet in order to be maintained at the bottom of the buffer tray. Inset shows a scheme representing a blade cutting sagittal slices. The brain is in gray and the hippocampus in green. The scheme was created in Biorender. (G) Overview of the water bath containing the storage chamber enclosing the brain slices. Tissue is maintained at a temperature of 34°C. (H) Top view of a storage chamber filled with sucrose solution. A gauze net maintains slices at ∼half-height of the beaker. A small micro filter candle supplies 95% O2 and 5% CO2 gas mixture to the solution. Very small gas bubbles are delivered by micro filter candles with a porosity of 4 corresponding to small pores. (I) Front view of the patch-clamp setup. The microscope is mounted on a table allowing movement in the two horizontal X-Y directions. Manipulators to move pipette are left and right to the recording table. An optical 4-time filter changer (Luigs&Neumann) containing magnification glasses is mounted on the top of the microscope and the digital camera (not appearing in the overview) on top of optic changer. (J) Recording chamber containing a brain slice. A water immersion objective (40X) is on top of the slice to visualize neurons. At the right a patch pipette in a pipette holder (Luigs&Neumann) with a bracket (Luigs&Neumann) surrounding the pipette holder to stabilize pipette. Inset shows the stainless steel slice hold-down flat frame with threads (Warner Instruments).

Article Snippet: Mount the slices on microscope slides (Superfrost slides, Fisher Scientific, Epredia J1800AMNZ) using mounting medium (Fluoromount aqueous medium, Sigma-Aldrich, Cat. No. F4680).

Techniques: Patch Clamp, Microscopy, Transferring, Slice Preparation