Hematoxylin Staining of Mammary Glands

P. Keller 2007

Hematoxylin Staining of Mammary Glands (adapted from Charlotte)

Day 1:

Remove mammary glands, fix in 10% neutral buffered formalin overnight

Day 2:

Prepare hematoxylin stain

Make the following solutions:

1. Stock hematoxylin (0.1 g hematoxylin in 1 ml 95% EtOH)

2. 1 N HCl (dilute 8.33 ml 12 N HCl in 91.67 ml MQ H2O)

3. 13.8 mM Ferric Chloride (FeCl3 6H20 FW 270.3 Sigma #F2877)

a. 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

1. Acetone: 1 hour

2. 100% EtOH: 1 hour

3. 95% EtOH: 1 hour to overnight

4. Incubate overnight in hematoxylin stain solution

Day 3:

Dehydrate tissues and mount for pictures

1. Running tap water: 1 hour

2. 70% EtOH: 1 hour

3. 95% EtOH: 1 hour

4. 100% EtOH 1: 1 hour

5. 100% EtOH 2: 1 hour

6. Xylene 1: 1 hour

7. 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