[Histonet] Acid Phosphatase Protocol

[Tony Henwood (SCHN)]

Hi April,
Try this

Control tissues: liver, kidney and prostate
Fixation and Sectioning: Air dried unfixed 8µm frozen sections

Solutions:

1.	Substrate solution
Naphtol AS-B1 phosphate		0.005g (Fluka 70494  1g)
Dimethylfomamide			0.5ml
Warning: Suspected Carcinogen – see MSDS

2.	Acetate buffer pH 4.8
Sodium acetate				4.8g 
Distilled water				100ml
0.6% Acetic Acid				14.75ml
pH to 4.8
Make up to 1000 ml with distilled water

3.	Sodium nitrite: 
Warning: Toxic – see MSDS
Sodium nitrite		 	1.0g
Distilled water			25.0ml

4.	Pararosaniline-HCl stock solution. 
Warning: Suspected Carcinogen – see MSDS
Pararosaniline hydrochloride 
(Basic Fuchsin CI 42510)	  	0.8g
Distilled water				16ml
Concentrated Hydrochloric acid		4ml
Heat gently, cool to room temperature and filter (store 4C)

5.	Incubation solution:
Take 0.4ml solution 3 + 0.4ml solution 4 and mix. Stand for 2 minutes and then add to:
Substrate Solution (Solution 1)		0.5ml
Acetate buffer pH 4.8			25ml
Adjust with 0.1M NaOH to pH 4.5-5.0.

Procedure:

1.	Take air-dried sections and incubate in the incubation solution at 37oC for 1 hour
2.	Wash with distilled water and counterstain with Harris's Haematoxylin for 1 minute.
3.	Wash in running tap water
4.	Dehydrate rapidly through fresh alcohols to xylene and mount.

Results:

Acid phosphatase activity		red
Nuclei					blue

Regards
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

________________________________________
From: Oler, April via Histonet [histonet at lists.utsouthwestern.edu]
Sent: Friday, January 26, 2018 6:26 AM
To: 'histonet at lists.utsouthwestern.edu'
Subject: [Histonet] Acid Phosphatase Protocol

Good afternoon,

I am writing to you all because I am in search of Acid Phosphatase muscle protocols that do not utilize sodium barbital. I'm hoping to eliminating sodium barbital from my lab in the near future. If anyone is willing to share their protocol, or advice, I would very much appreciate any assistance.

Thank you!

April Oler, HT(ASCP)
Senior Clinical Technologist
IPOX/DIF/Muscle Laboratories
Michigan Medicine
Ann Arbor, MI




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