H&E Staining of Mouse Tissue

Materials

• xylene/Citrisolv
• 100% Ethanol
• 70% Ethanol
• H2O
• Mayer's Hematoxylin
• 1% Eosin Y (in 95% ethanol)
• 95% Ethanol

Protocol

• Removing Wax and Rehydrating

1. Place slide on edge and bake for 15 min at 60 degree C to melt wax
2. Deparaffinize and rehydrate sections
   1. Immerse for 5 min with agitation in xylene/Citrisolv
   2. Immerse for 5 min with agitation in 100% ethanol
   3. Immerse for 5 min with agitation in 70% ethanol
   4. Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min

• H&E staining

1. Dip slide into Mayer's Hematoxylin and agitate for 30 sec
2. Rinse slide in H2O for 1 minute
   1. depending on the intensity desired, repeat this step if necessary
3. Dip slide in 1% eosin Y fo 10-30 sec with agitation
   1. For adipose tissue, 20 seconds allows for proper staining.

• Dehydration

1. Place slides in 95% Ethanol two times for 30 seconds each.
2. Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
3. Wash twice with fresh xylene/Citrisolv for 30 seconds.
4. Let slide dry.
5. Place 1-2 drops of mounting medium on slide and place coverslip over
6. Seal coverslip with sealant(nailpolish)

Notes

• Be sure to shake Eosin prior to placing slide in for staining because it will settle.
• The staining will get more intense after time, be sure not to oversaturate section
• Especially during the summer months, be sure to not let the slides set out overnight/extended period of time.

• Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986