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

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
50-ml centrifuge tube by Falcon	VWR	734-0453
Formaldehyde solution, 37%	Carl Roth	CP10.2	Acid-free, stabilized with ~10% MeOH
Glacial acetic acid	Alfa Aesar	36289.AP
Eosin Y disodium salt	Sigma-Aldrich	E4382	Certified by Biological Stain Commission
Phosphate Buffered Saline (PBS)	Merck	L1825	Dulbecco's formualtion, w/o calcium and magnesium
Sample Tubes by Nalgene	Carl Roth	ATK5.1
Rocking Shaker ST5	CAT	60281-0000
Cellulose tissue paper	VWR	115-0600
Microcentrifuge tubes by Eppendorf	VWR	211-2120	Safe-lock, 2.0 ml
Ethanol absolute by Baker Analyzed	VWR	80252500
Petri dish by Sterilin
Forceps, by USBECK Laborgeräte	VWR	232-0096
Plastic pasteur pipette	Carl Roth	EA68.1	Graduated, 1 ml