Eosin Staining of Soft-Tissue Samples: An Eosin-Based Procedure for Whole Mouse Kidney Staining to Visualize Specific Tissue Microstructures

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

1. Eosin staining protocol

1. To fixate soft-tissue samples, fill a 50-mL centrifuge tube with a fixative solution containing 9.5 mL of 4% (v/v) formaldehyde solution (FA) and 0.5 mL of glacial acetic acid (AA).
   NOTE: Prepare the FA solution freshly from a 37% acid free FA solution stabilized with approximate 10% methanol. Dilute the FA solution further with Dulbecco's phosphate buffered saline (DPBS). Choose DPBS without calcium and magnesium. Keep the dilute FA solution no longer than one month. During acidification the pH of the fixative solution is changing from neutral to approximately 3.
   CAUTION: Because FA is acute organ toxic, corrosive and carcinogenic, the use of a fume hood is mandatory and appropriate protective personal equipment must be used.
   1. Add the freshly removed soft-tissue sample to a 50-mL centrifuge tube and refrigerate the 50-mL centrifuge tube for 24-72h.
      NOTE: The protocol can be paused here.
   2. Wash the soft-tissue sample with DPBS solution for 1h.
2. To stain the fixated soft-tissue sample (e.g., a whole mouse kidney), place the soft-tissue in 2 mL of eosin Y-staining solution and incubate the sample for 24 h. Keep the sample on a horizontal shaking plate for a smooth rocking (ca. 60 rpm) during the incubation process.
   NOTE: The eosin Y-staining solution has a concentration of 30% (w/v) in distilled water. Choose the volume of the staining solution in such a way that the sample is completely covered by the staining solution and allow the sample to move freely within the sample container. The incubation time may differ for other samples and must be adjusted accordingly.
3. After staining, remove the soft-tissue sample carefully from the sample container.
   1. Carefully remove excess of staining agent with cellulose tissue paper.
   2. Place the soft-tissue sample in a conical sample container above an ethanol vapor phase for storage and further use.
      NOTE: The conical sample container must always contain a few drops of 70% (v/v) ethanol at the bottom of the tube to keep the soft-tissue sample moist and prevent artifacts.