[Histonet] sodium 5.5-diehylbarbiturate
Tony Henwood
AnthonyH <@t> chw.edu.au
Sun Mar 1 16:10:10 CST 2009
Lazo,
This is our technique:
Adenosine Triphosphatase (ATPases)
Use
Demonstration of muscle fibre types.
Underlying Principle
The principle relies on the ability of the enzyme to remove the terminal phosphate from the ATP, which then combines with calcium in the incubation solution to form an insoluble calcium phosphate. Cobalt is then exchanged for the calcium, which, after reaction with ammonium sulphide, forms a black, insoluble cobalt sulphide at the site of enzyme activity.
Fixation and Sectioning
Air dried unfixed 8µm cryostat sections
Reagents
1. Acid Pre-incubation medium
0.2M Sodium Acetate
Sodium Acetate Anhydrous 0.82 g
Distilled water 50 ml
0.2M Acetic Acid
Glacial Acetic Acid 0.6 ml
Distilled water 50 ml
0.2M Acetate Buffer:
pH 4.3 pH 4.6
0.2M Sodium Acetate 11 ml 18 ml
0.2M Acetic Acid 12 ml 13 ml
Adjust to pH 4.3 or 4.6 with 0.2M Sodium Acetate or 0.2M Acetic Acid
2. 7.5% Calcium Chloride
Calcium Chloride 7.5g
Distilled water 100ml
3. 7.05% Glycine
Glycine 7.05g
Distilled water 100ml
Aliquot 2ml into tubes labeled "G" and store at -20oC
4. 5.625% Sodium Chloride
Sodium Chloride 5.625g
Distilled water 100ml
5. 3.5% Sodim Hydroxide
Sodium Hydroxide 3.5g
Distilled water 100ml
6. Alkaline Stock
Warning: Irritant - see MSDS
7.05% Glycine 2ml
7.5% Calcium Chloride 2ml
5.625% Sodium Chloride 2ml
3.5% Sodium Hydroxide 2ml
Distilled water 42ml
7. Incubating Medium
1. Prepare fresh Alkaline Stock for each run
2. pH close to 11 and place in 37oC Oven for 20minutes
3. pH to 11 using 0.1M NaOH (to activate the ATP)
4. Add 0.04g ATP (Sigma A-7699)
5. pH to 9.35 using 0.1M HCl
8. 1% calcium chloride
Calcium chloride 5.0 g
Distilled water 500ml
9. 2% Cobalt chloride
Warning: Suspected Carcinogen - see MSDS
Cobalt chloride 10.0 g
Distilled water 500 ml
10. 1% Ammonium sulphide
Warning: Flammable liquid, Irritant, Toxic stench - see MSDS
20% ammonium sulphide 0.5 ml
Distilled water 9.5 ml
Staining Method
1. Place one slide in 4.3 and one in 4.6 buffer at room temperature for 20 minutes
2. Wash each in distilled water 3 times
3. Place these slides (one from the 4.3 solution , one from 4.6 solution and the 9.4 slide) in the 9.4 substrate at 37°C for
pH 9.4 10 mins
pH 4.6 30 mins
pH 4.3 45 mins
4. Place slides in 2 changes of 1% calcium chloride 3 min each
5. Place slides in 2% cobalt chloride 3 min
6. Wash well in distilled water
7. In fume cupboard drain slides well and place in 1% ammonium sulphide solution for 1 min
8. Wash well in tap water
9. Dehydrate clear and mount.
Results
pH 9.4
Type 1 fibres pale
Type 2A fibres intermediate
Type 2B fibres dark
pH 4.3 and pH 4.6
pH 4.3 pH 4.6
Type 1 fibres dark dark
Type 2A fibres pale pale
Type 2B fibres pale intermediate
Type 2C fibres intermediate dark
Notes
This is a complicated stain and there are several areas in which one needs to be careful in order to achieve a good fibre type differentiation.
1. The pH of all solutions is critical
2. Timing is crucial
1. The stock ammonium sulfide must still be yellow. As it ages or oxidizes, it becomes more red and cannot be used.
2. All solutions must be adjusted at the temperature they will be used.
Difficulties with ATPase technique (1)
Problem
Possible Causes
Remedies
Poor fibre type differentiation
1. Inaccurate pH reading.
2. Severe pathological condition.
1. Carefully calibrate pH meter using standard solutions of known pH.
2. Assess fibre typing using pH 4.3/4.6 ATPase sections
Very pale ATPase reaction
1. Incubation time too short
2. pH of incubating medium too low
3. Incubation temperature < 37oC
4. Ammonium sulfide solution has turned deep yellow or brown due to oxidation.
1. Incubate sections longer
2. Carefully calibrate pH meter
3. Check temperature of incubator
4. Ensure cap on ammonium sulfide solution is replaced tightly after use
Very dark ATPase reaction
1. Over incubation
2. pH too high
1. Reduce incubation time
2. Carefully calibrate pH meter
Artefact patches within muscle fibres
1. Dirt or grease on glass slides
2. Fingerprint on slide under sections
1. Ensure clean slides are used
2. Pick up glass slides at the opposite end to where the section is to be positioned
References
1. Loughlin, M. (1993). Muscle biopsy. A laboratory investigation. Butterworth-Heinemann pp.78-79.
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lazo Pendovski
Sent: Saturday, 28 February 2009 12:59 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] sodium 5.5-diehylbarbiturate
Hi,
I need help with ATPase muscle fiber staining in lambs. I am investigating the skeletal muscle fibers in lambs breaded extensively in mount-hills regions in Macedonia.
But, I have a problem with a one chemical - sodium 5.5-diehylbarbiturate. The problem is that I can't get this substance in my country.
So please, does any one know substitute or a protocol without Sodium barbital?
Any help you can give me would be greatly appreciated.
Lazo
ass. m-r Lazo Pendovski, M.Sc., DVM
University of St. "Cyril and Methodist"
Faculty of Veterinary medicine
Department of Functional Morphology
Lazar Pop- Trajkov 5-7
1000 Skopje, R. of Macedonia
tel: +389 2 3240 710
fax: +389 2 3114 619
mob:+389 70 766 017
e-mail: lpendovski <@t> fvm.ukim.edu.mk _______________________________________________
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