Vibratome Sectioning of Whole Murine Kidney: A Technique to Generate Thin Sections of Murine Kidney

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Prepare Solutions

1. Prepare 1 L of Krebs-Ringer Bicarbonate (KRB) solution containing 120.35 mM NaCl, 5.9 mM KCl, 15.5 mM NaHCO₃, 1.2 mM Na₂HPO₄,1.2 mM MgCl₂, 11.5 mM glucose, and 2.5 mM CaCl₂. On the day of use, maintain the KRB solution on ice.
   NOTE: KRB can be stored at 4 °C for up to one week and should be pre-bubbled with a mix of 97% O₂ and 3% CO₂ for at least 10 min prior to use.

2. Vibratome Sectioning of the Kidney

1. Use blunt-ended forceps to gently grasp and remove the prepared kidney from the ice-cold KRB solution. Immediately place the kidney on absorbent paper for ~2–4 s to remove excess external moisture. Gently roll the kidney across the absorbent paper to ensure that all sides of the parenchyma have dried so that there is optimal adhesion of the kidney to the vibratome stage.
   NOTE: As the renal pelvis is located inside the kidney and therefore protected by the outer parenchyma, this short drying period would not be detrimental to tissue integrity.
2. Immediately apply a thin layer of cyanoacrylate glue (~1 cm²) to the base of the vibratome specimen plate and use blunt-ended forceps to place the kidney, ureter side down, on the area covered in glue. Gently apply downward pressure to the top of the kidney with the flat edge of the forceps for approximately 10–20 s to dry the glue.
   NOTE: To stabilize the kidney during this procedure, use an additional pair of forceps to keep the kidney upright as the glue dries. It is critical that the kidney adheres to the specimen plate in an upright position so that sections are cut straight. To ensure that the kidney has successfully adhered to the specimen plate, gently push the side of the kidney. If the kidney has successfully adhered to the plate, the base of the kidney should stay secured to the plate.
3. Firmly secure the specimen plate to the bottom of the buffer tray. Adjust the level of KRB solution so that the top of the kidney is fully immersed. During sectioning steps, as the vibratome blade moves deeper into the buffer tray, remove KRB solution so that the blade holder does not become immersed in solution.
   NOTE: If the glue has not completely dried before proceeding with this step, the glue will often move up from the base and onto the kidney parenchyma. This excess glue will make sectioning more variable. If this does happen, remove the kidney from the plate and proceed with a fresh kidney preparation.
4. For automatic vibratome sectioning, select the start and end positions of the vibratome blade-cutting cycle. Verify these positions are ~0.5–1 cm clear of the kidney to ensure that with each blade advancement, the entire kidney plane is sectioned.
5. Prepare a multi-well plate (24- or 48-well) by filling wells with KRB solution, and place the plate on ice.
   NOTE: When generated, individual sections should be placed into separate wells to keep track of section depth.
6. Start the automatic cutting process. During the initial pass of the blade, ensure that the blade makes contact with the very top of the kidney. If contact is not made, adjust the starting Z-position of the blade.
7. Using forceps, collect sections that are liberated from the kidney. Immediately transfer the sections to individual wells and note the Z-depth of the sections to gauge the approximate PKJ location within kidney sections.
   NOTE: Depending on the cut parameters, some sections may not be cut free from the kidney block. If this occurs, carefully use fine spring scissors to cut sections from the kidney block. Users are also encouraged to actively visualize sections under a light microscope whilst free-floating in individual wells to ensure optimal cut settings and tissue location. Sections containing the PKJ will typically be derived ~ 1000–1500 µm from the top of the kidney.
8. Continue the sectioning protocol until the PKJ regions become more apparent. Refer to the representative results section for a description of the PKJ regions as sectioning proceeds.
9. At this point, optimize the sectioning parameters to ensure that the sections are liberated from the kidney block uniformly and are intact. Additionally, make sure that the PKJ regions are continuous and unbroken because broken PKJ walls will not allow adequate imaging of cells within the wall due to collapse. If walls become broken, decrease the sectioning speed and increase the section thickness and continue to observe sections under a light microscope to fine-tune the cutting parameters.
10. Store sections at 4°C in KRB solution until experimentation begins.