Difference between revisions of "H&E Staining of Mouse Tissue"
From Bridges Lab Protocols
(Created page with "==Materials== *xylene *100% Ethanol *70% Ethanol *H2O *Mayer's Hematoxylin *1% Eosin Y (in water) *95% Ethanol ==Protocol== *Removing Wax and Rehydrating # Place slide on ed...") |
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| Line 1: | Line 1: | ||
==Materials== | ==Materials== | ||
| − | *xylene | + | *xylene/Citrisolv |
*100% Ethanol | *100% Ethanol | ||
*70% Ethanol | *70% Ethanol | ||
*H2O | *H2O | ||
*Mayer's Hematoxylin | *Mayer's Hematoxylin | ||
| − | *1% Eosin Y (in | + | *1% Eosin Y (in 95% ethanol) |
*95% Ethanol | *95% Ethanol | ||
| 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 21: | Line 21: | ||
##depending on the intensity desired, repeat this step if necessary | ##depending on the intensity desired, repeat this step if necessary | ||
#Dip slide in 1% eosin Y fo 10-30 sec with agitation | #Dip slide in 1% eosin Y fo 10-30 sec with agitation | ||
| − | #Wash twice with fresh xylene for 30 seconds. | + | ##For adipose tissue, 20 seconds allows for proper staining. |
| + | *Dehydration | ||
| + | #Place slides in 95% Ethanol two times for 30 seconds each. | ||
| + | #Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol. | ||
| + | #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 | ||
#Seal coverslip with sealant(nailpolish) | #Seal coverslip with sealant(nailpolish) | ||
| − | |||
==Notes== | ==Notes== | ||
*Be sure to shake Eosin prior to placing slide in for staining because it will settle. | *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 | *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 | *Protocol take from summary of CSH Protocols; 2008; doi:10.1101/pdb.prot4987 and CSH Protocols; 2008; doi:10.1101/pdb.prot4986 | ||
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
- Place slide on edge and bake for 15 min at 60 degree C to melt wax
- Deparaffinize and rehydrate sections
- 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 70% ethanol
- Place slide(s) in appropriate buffer (For H&E, use H2O) for 1 min
- H&E staining
- Dip slide into Mayer's Hematoxylin and agitate for 30 sec
- Rinse slide in H2O for 1 minute
- depending on the intensity desired, repeat this step if necessary
- Dip slide in 1% eosin Y fo 10-30 sec with agitation
- For adipose tissue, 20 seconds allows for proper staining.
- Dehydration
- Place slides in 95% Ethanol two times for 30 seconds each.
- Transfer slides into 100% Ethanol for 30 seconds, repeat with fresh 100% Ethanol.
- Wash twice with fresh xylene/Citrisolv for 30 seconds.
- Let slide dry.
- Place 1-2 drops of mounting medium on slide and place coverslip over
- 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
