Day 1
Remove mammary glands, fix in 10% neutral buffered formalin overnight
Day 2
Prepare hematoxylin stain
Make the following solutions:
- Stock hematoxylin (0.1 g hematoxylin in 1 ml 95% EtOH)
- 1 N HCl (dilute 8.33 ml 12 N HCl in 91.67 ml MQ H2O)
- 13.8 mM Ferric Chloride (FeCl3 6H20 FW 270.3 Sigma #F2877)
- dissolve 0.112 g ferric chloride hexahydrate in 30 ml MQ H2O
Mix the reagents in the order listed below:
0.49 ml stock hematoxylin solution
35 ml 95% EtOH
15 ml 13.8 mM FeCl sol’n
10 ml 1 N HCl
100 ml 95% EtOH
Allow to stand for 2-24 hours, use within 1 week
Defat the tissue through graded organics/alcohols then stain
- Acetone: 1 hour
- 100% EtOH: 1 hour
- 95% EtOH: 1 hour to overnight
- Incubate overnight in hematoxylin stain solution
Day 3
Dehydrate tissues and mount for pictures
- Running tap water: 1 hour
- 70% EtOH: 1 hour
- 95% EtOH: 1 hour
- 100% EtOH 1: 1 hour
- 100% EtOH 2: 1 hour
- Xylene 1: 1 hour
- Xylene 2: I hour to overnight
Mount glands in permount on slides for pictures
Alternatively, store in methyl salicylate and press between slides for pictures
For histology, incubate slides in xylenes to release glands from permount, and reverse steps 2-6, submit to histology for paraffin embedding, sectioning and HE staining
