Difference between revisions of "H&E Staining of Mouse Tissue"

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(Materials)
(Protocol)
 
Line 12: Line 12:
 
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
 
# Place slide on edge and bake for 15 min at 60 degree C to melt wax
 
# Deparaffinize and rehydrate sections
 
# Deparaffinize and rehydrate sections
## Immerse for 5 min with agitation in xylene
+
## Immerse for 5 min with agitation in xylene/Citrisolv
 
## Immerse for 5 min with agitation in 100% ethanol
 
## Immerse for 5 min with agitation in 100% ethanol
 
## Immerse for 5 min with agitation in 70% ethanol
 
## Immerse for 5 min with agitation in 70% ethanol
Line 25: Line 25:
 
#Place slides in 95% Ethanol two times for 30 seconds each.
 
#Place slides in 95% Ethanol two times for 30 seconds each.
 
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
 
#Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
#Wash twice with fresh xylene for 30 seconds.
+
#Wash twice with fresh xylene/Citrisolv for 30 seconds.
 
#Let slide dry.
 
#Let slide dry.
 
#Place 1-2 drops of mounting medium on slide and place coverslip over
 
#Place 1-2 drops of mounting medium on slide and place coverslip over

Latest revision as of 14:51, 9 June 2015

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