Jun 01, 2020

Public workspaceVerhoeff’s Iron Hematoxylin Staining

DOI
dx.doi.org/10.17504/protocols.io.be9sjh6e
  • Jeff Z. Chen1,
  • Deborah A. Howatt1,
  • Michael K. Franklin1,
  • Jessica J. Moorleghen1,
  • Hisashi Sawada1,
  • Hong S. Lu1,
  • Alan Daugherty1
  • 1University of Kentucky
Jeff Z Chen
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DOI: dx.doi.org/10.17504/protocols.io.be9sjh6e
External link: http://cvrc.med.uky.edu/lab-protocols
Protocol Citation: Jeff Z. Chen, Deborah A. Howatt, Michael K. Franklin, Jessica J. Moorleghen, Hisashi Sawada, Hong S. Lu, Alan Daugherty 2020. Verhoeff’s Iron Hematoxylin Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.be9sjh6e
Manuscript citation:
Chen JZ, Sawada H, Moorleghen JJ, Weiland M, Daugherty A, Sheppard MB. Aortic Strain Correlates with Elastin Fragmentation in Fibrillin-1 Hypomorphic Mice. Circ Rep. 2019;1(5):199–205. doi:10.1253/circrep.CR-18-0012
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 17, 2020
Last Modified: June 01, 2020
Protocol Integer ID: 35858
Keywords: aorta, elastin, histology, verhoeff,
Abstract
This protocol is used to stain elastic tissue.
This results in elastic fibers and nuclei stained black.
Staining of elasin allows for quantification of elastin breaks.
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Verhoeff's Iron Hema...
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Materials
1. Verhoeff’s iodine (50 ml stock solution)
Iodine - 1 g
Potassium iodide - 2 g
Distilled water - 50 ml

2. Verhoeff’s iron hematoxylin (36 ml)
Hematoxylin (5% wt/vol) in absolute alcohol - 20 ml
Ferric chloride (10% wt/vol) - 8 ml
Verhoeff’s iodine - 8 ml
Mix a fresh solution, in the order given for each usage.

3. Ferric chloride (2% wt/vol)

4. Alcohol (95% vol/vol)

Acetone Fischer S25120A
Iodine Fischer I35-100
Potassium Iodide Sigma P8256
Hematoxylin Sigma H9627
Ferric (III) Chloride Sigma F2877
100% ethanol Fischer BP2818-4
Xylene Millipore XX0060-4

Before start
Place slides in glass holder. Be sure that slides are labeled appropriately.
Prepare reagents before starting.
Elastin Staining
Elastin Staining
45m 30s
45m 30s
Acetone to fix
Note
If slides keep falling off slide, air dry for 30 mintues before fixation

5m
Water (wash)
Verhoeff’s iron hematoxylin
20m
Water (3 washes)
Ferric chloride (2% wt/vol)

Water 3 washes
Check slide for differention at 1m30s and for every 30s afterward


Note
* Halt staining when nuclei and elastin fibers are still black with weak background.

*** If over-differentiated, go back to the step 3.

1m 30s
Ethanol (95% vol/vol)
30s
Ethanol (100% vol/vol)
30s
Xylene
3m
Air dry uncovered
15m
Permount and coverslip