Aniline Blue Staining Protocol
1) Bring samples up to 100% ethanol
2) When trying to remove the waxy cuticle, move through histoclear series
-
25% histoclear + 75% 100% ethanol for 1 hour
50% histoclear + 50% 100% ethanol for 1 hour
75% histoclear + 25% 100% ethanol for 1 hour
100% histoclear for 1 hour
100% histoclear for 1 hour
3) Bring back into ethanol
-
75% histoclear + 25% 100% ethanol for 1 hour
50% histoclear + 50% 100% ethanol for 1 hour
25% histoclear + 75% 100% ethanol for 1 hour
100% ethanol for 1 hour
100% ethanol for 1 hour
4) Rehydrate samples in water
-
25% water + 75% 100% ethanol for 1 hour
50% water + 50% 100% ethanol for 1 hour
75% water + 25% 100% ethanol for 1 hour
100% water for 1 hour
100% water for 1 hour
5) If vacuum infiltrating, pull vacuum for 10 minutes in 1:10 dilution of aniline blue and 1x
PBS buffer. Replace with new mixture, and repeat 10 minutes.
6) Let tissue sit in stain overnight (or as long as necessary) in 1:10 dilution of aniline blue
and 1x PBS buffer
For 60 mLs
25/75 = 15 mLs +45 mLs
50/50 = 30 mLs + 30 mLs
75/25 = 45 mLs + 15 mLs
1X PBS buffer
8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
Dissolve reagents in 800 mLs H20. Adjust pH to 7.4 with HCl, add more H20 to 1 L.
Autoclave