[Histonet] H + E Staining protocols

[Lucas Olson]

Dear All,

There are many variants to the standard H + E protocol. I'm seeking to
optimize the current protocol our laboratory uses (pasted below). Do you
guys have any suggestions of optimization (e.g. cutting down on the long
water rinse after hematoxylin and increasing bluing reagent time)? Or if
you have protocols that you'd think would be better please let me know. Our
lab deals primarily with bone/cartilage/muscle.

Our current protocol:
Staining Preparation
1. After sectioning samples, dry in oven.
Time Temp C
Overnight 37
2-3 hours         45
30 minutes 55

Deparaffinization
1. Remove paraffin with xylene
a. 2 minutes xylene
b. 2 minutes in fresh xylene
c. 2 minutes in fresh xylene
d. 2 minutes in fresh xylene
2. Remove excess xylene
3. Rehydrate samples
a. 1 minute absolute EtOH
b. 1 minute different bucket of absolute EtOH
c. 30 seconds in 95% absolute EtOH
d. 45 seconds in 70% EtOH
4. Rinse with water for 1 minute
Hematoxylin and Eosin Staining
1. Stain in hematoxylin (VWR 95057-858) for 5-10 minutes
a. Optimized time for calvarium bone: 10 minutes
b. Solution may be filtered to remove oxidized particles
2. Rinse in running tap water for 20 minutes
3. Decolorize in Clarifier 1 (1 second)
a. Longer time (up to 3 seconds) will yield a lighter color. Discard after
each use.
4. Immerse in VWR Bluing Reagent (30 sec- 1 minute)
a. Dilute 25mL bluing reagent in 475 mL ultrapure H2O to working stock
b. Optimized time for calvarium bone: 90 seconds
5. Rinse well in tap water (5 minutes)
6. Rinse in 95% ETOH (30 sec- 1 minute)
7. Counterstain in Eosin (30 sec- 90 sec)
a. Optimized time for calvarium bone: 30 seconds
8. Dehydrate
a. ETOH 95% (3 minutes)
b. ETOH 95% (3 minutes)
c. ETOH 100% (3 minutes)
d. ETOH 100% (3 minutes)
9. Clear in Xylene (5 minutes)
10. Clear in new Xylene (5 minutes)
11. Mount with Cytoseal in fume hood and coverslip




Thanks!
Lucas Olson