[Histonet] pituitary stain

Houston, Ronald Ronald.Houston <@t> nationwidechildrens.org
Fri Oct 22 09:23:38 CDT 2010


The University of Nottingham Histology department has an extensive website of histology protocols:


There is a protocol for PAS/OG on that site. I do not have any experience using it, but as it comes out of John Bancroft's old lab it will undoubtedly be very sound. It does differ somewhat from the method of AGE Pearse that I followed many, many moons ago in Glasgow. We also always performed an OFG (orange-fuchsin-green) method on all pituitary bxs (if you need a copy of that let me know).As I'm sure you are aware, tinctorial stains for pituitary adenomas have been replaced with immunohistochemistry for the hormones ACTH, FSH, GH, LH, prolactin and TSH:


1.      Section to 70% alcohol

2.      leave in 70% alcohol for 5 minutes

3.      transfer to  Solution "A" for 15 minutes at room temp

4.      Wash in 70% alcohol for 5 minutes

5.      transfer to reducing rinse "B" for 5 minutes

6.      70% alcohol for 3 minutes

7.      rinse in running tap water

8.      Schiff's reagent 20-30 minutes

9.      wash in water 3 minutes

10.  Celestin Blue solution      10 minutes

11.  Rinse well in water

12.  Hematoxylin     10 minutes

13.  wash in water

14.  2% Orange G in 5% phosphotungstic acid for 3 minutes

15.  wash in water

16.  dehydrate, clear and mount


Nuclei         -     black

RBC's          -     yellow-orange

Alpha granules -     orange

Beta granules  -     magenta (PAS +ve)

Solution "A"

   Periodic acid     -     400mg

   Distilled water   -     15 ml

   100% alcohol      -     35 ml

Reducing Solution "B"

   Sodium thiosulphate     -     1gm

   Distilled water         -     20ml

   100% alcohol            -     30ml

   HCl                     -     0.5ml


PAS converts 1,2 glycols to aldehyde. The reducing step is thought to block the subsequent reaction of collagen and retic fibers with the Schiff's reagent. Beta granules contain muco- or glycoproteins that react with Schiff's to give their magenta staining. Orange G, which is an acidic dye, stains the alpha granules. As RBC's are also acidophilic in nature, they also pick up the orange coloration.


Pearse AGE. The cytochemical demonstration of gonadotrophic hormone in the human anterior hypophysis. J Pathol Bacteriol. 1949: 61; 195-202

If you need a recipe for the celestin blue solution, drop me a line and I'll get it to you, but you should find it in most good histotechnology text-books, or on-line

Best wishes

Ronnie Houston, MS HT(ASCP)QIHC
Anatomic Pathology Manager

ChildLab, a Division of Nationwide Children's Hospital

700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.houston <@t> nationwidechildrens.org<mailto:ronald.houston <@t> nationwidechildrens.org>

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of mohamed abd el razik
Sent: Friday, October 22, 2010 8:36 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] pituitary stain


dear all

can you share me pituitary stain protocol called

PAS/ celestine blue / Orange G?

thanks in advance



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