[Histonet] JB4 embedded specimens

Tony Henwood (SCHN) tony.henwood at health.nsw.gov.au
Thu Sep 24 19:03:23 CDT 2020

I have found that that this gives a pleasing tinctorial result:

Haematoxylin and Eosin


Traditional HE procedures generally worked poorly on GM sections, with background staining of the plastic embedment and the loosening of sections from glass slides when alcoholic eosin is used. Aqueous solutions tend to stain poorly.

Sections are firstly stained with Celestine blue, followed by a regressive Harris's haematoxylin (excess stain is removed from the plastic with acid alcohol). The sections are then counterstained with buffered eosin Y/Phloxine B.


1.   Celestine Blue - Iron Alum
2.   Harris's Haematoxylin
3.   Buffer for Eosin Y/Phloxine B.
          Solution A    2.875ml Glacial Acetic acid in 500ml distilled water.
          Solution B    4.1g Sodium Acetate in 500ml distilled water.

          150 ml Solution A + 350 ml Solution B. pH to 4.85.

4.   Eosin Y/Phloxine B.
          Eosin Y (CI 45380)            5g.
          Phloxine B (CI 45410)     0.5g.
          Buffer                        500ml.
          Add 2 crystals Thymol.


1.	Hydrate sections.
2.	Stain in Celestine Blue 15 minutes.
3.	Wash in running water.
4.	Stain in Harris's Haematoxylin 15 minutes.
5.	Wash, differentiate and blue till surrounding plastic is clear.
6.	Stain in buffered Eosin for 6 minutes.
7.	Wash lightly in running water.
8.	Differentiate in alcohol till surrounding plastic is clear.
9.	Clear in Xylene and mount.


Castro,M.D., (1985) "A Haematoxylin - Eosin Phloxine Stain for tissues embedded in Glycol Methacrylate" J. Histotechn 8(1); 23-24.

Green,G.H.,Kurnein,F., (1981)  "Glycol Methacrylate embedding in General Histopathology " ACP Broadsheet 97.

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: Keena Leon Guerrero via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Friday, 25 September 2020 8:47 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] JB4 embedded specimens

Hello All,

I was wondering if anyone has a good Hematoxylin and Eosin (H&E) staining protocol for JB4 embedded specimens?


Keena Leon Guerrero
Histonet mailing list
Histonet at lists.utsouthwestern.edu

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