This article describes a detailed protocol for preparing thin sections of mouse kidney tissue using a vibratome. The method emphasizes maintaining tissue morphology while achieving uniform sectioning for downstream analysis.
To prepare thin sections of a mouse kidney, first, obtain a freshly harvested mouse kidney. Transfer the kidney onto a filter paper and dry it to ensure optimal adhesion to the vibratome specimen holder.
Next, mount the kidney, ureter side down, using a thin layer of cyanoacrylate glue applied to the base of the vibratome specimen holder, securing the kidney in an upright position. Transfer the specimen holder onto a buffer tray.
Fill the tray with a suitable buffer to fully immerse the top surface of the kidney. An ice bath surrounds the tray to prevent tissue damage from heat buildup during slicing. Set the vibratome blade to an appropriate angle, speed, and amplitude. Adjust the thickness parameter to obtain tissue sections of desired thickness.
During operation, the blade vibrates laterally and advances, penetrating through the tissue specimen. The vibrating blade allows the sectioning with less pressure and minimizes the tissue deformation, generating uniform kidney tissue sections of interest while maintaining the tissue morphology.
Meanwhile, assemble a multi-well plate containing a suitable chilled buffer solution to mimic the physiological conditions of the tissue. Now, transfer the sliced tissue sections into the buffer. Finally, store the tissue sections at low temperature until further downstream analysis.
Use blunt-ended forceps to gently grasp and remove the prepared kidney from ice-cold KRB solution. Immediately place the kidney on absorbent paper for approximately 2 to 4 seconds 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.
Immediately apply a thin layer of cyanoacrylate glue 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 to 20 seconds to dry the glue.
Firmly secure the specimen plate to the bottom of the buffer tray and adjust the level of KRB solution so that the top of the kidney is fully immersed. For automatic vibratome sectioning, select the start and end positions of the vibratome blade cutting cycle, 0.5 to 1 centimeter clear of the kidney, to ensure that the entire kidney plane is getting sectioned.
Start the automatic cutting process, making sure that the blade makes contact with the kidney. Using forceps, collect sections that are liberated from the kidney and immediately transfer them to individual wells.
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