Hematoxylin and Eosin Staining Protocol

Modified from UNC Histopathology Protocol

Updated 9/12/03 Reggie Hill
1. Deparaini!e in "ylene # and ## and ### $% min&tes'
2. Re(ydrate
a) Et*H 100+ $3 min&tes'
,) Et*H 100+ $3 min&tes'
c) Et*H 9%+ $3 min&tes'
d) Et*H 9%+ $3 min&tes'
e) Et*H -0+ $3 min&tes'
3. Rinse in distilled .ater $% min&tes'
4. Stain in (ematoxylin $/ min&tes' Filter before each use to remove oxidized particles
5. Rinse in r&nning tap .ater $20 min&tes'
6. Decolori!e in acid alco(ol $1 second' Can go up to 3 seconds. Longer =Lighter Discard after each use
7. Rinse .ell in tap .ater $% min&tes'
8. #mmerse in 0it(i&m 1ar,onate $3 Seconds' Longer time = floating tissue
9. Rinse in tap .ater $% min&tes'
1. 1o&nterstain in Eosin $1% seconds'
11. De(ydrate
a) Et*H 9% + $3 min&tes' !iscard after each use
,) Et*H 9%+ $3 min&tes'
c) Et*H 100 + $3 min&tes'
d) Et*H 100 + $3 min&tes'
12. 1lear in "ylene # and ## $% min&tes'
13. 2o&nt .it( 1ytoseal in &me (ood)
Stock Solutions EOSIN:
Stoc3 1+ a4&eo&s Eosin56
Stoc3 1+ a4&eo&s P(loxin 7
Working Solutions Eosin:
100ml stoc3 Eosin
10 ml stoc3 P(loxin 7
-80 ml 9%+ Et(anol
9 ml glacial :cetic :cid
Working Solution: - Hematoxylin
Harris Hematoxylin; Sigma; HHS532; 1 0iter
Working Solution: - Lithium Carbonate !"#$
0it(i&m 1ar,onate; 9-g
dH
2
*; 3%00 ml
Working Solution: - %!&'$ (ci) (lcohol
9%+ Et(anol; 2%-8 ml
dH
2
*; 9%0ml
H10; 9ml