RE: Frozen Muscle Stains

From:Tony Henwood

Answers as follows

Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

-----Original Message-----
From: []
Sent: Tuesday, 22 April 2003 9:11
Subject: Frozen Muscle STains

Hi Everyone, Thanks for the responses, I appreciate it. Heres my questions: 

 " how long does a frozen muscle have to be in the -70 freezer before you can cut it on the cryostat?  " 
**** Doesn't have to be at -70oC for a specific period of time. Have cut and stained frozen sections immediately after freezing as well as several months at -70oC
Do any of you use control slides when staining? And if you do use controls do they have to be fresh tissue or paraffin embedded tissue?  
    No use inbuilt controls 

The stain giving us the most problems is the ATP. We can't seem to get it to work consistently. We also are not doing the pH, and wonder how important that is to the staining process? I'm thinking pretty important.  
Look at the following site - this technique works brilliantly:
 Lowe, James. Histology Lab: Adenosine Triphosphatase (ATPase). Http://

The staining protocol we are using is as follows:
1. Slides in Sodium barbital buffer for 10 minutes.
2. incubate slides in incubation solution at 37 degrees c. for 30 minutes.
3. Transfer slides to 2% cobaltous chloride solution for 3 minutes.
4. Wash slides in distilled water for 5 minutes.
5. we then dip slides (one by one) in Ammonium sulfide 6-7 times, sometimes more. I believe the stain gets darker the more you dip.
6.Rinse in water and check differentiation. At this Point I expect to see two different shades of brown.
7. Wash in running tap water for 20 minutes, if everything looks like it should.
8.Deydrate in graded alcohols, clear in histoclear or xylene and coverslip in synthetic mounting medium.

The solutions are made the following way:

Sodium barbital buffer:
sodium barbital 2.062g
calcium chloride .999g
d. water 1000 ml
The ph is then supposed to be adjusted to 9.4. Stable for 2 months. Store in refrigerator.
We do not do anything with the pH because this is not how we were taught to do the stain.

2% Cobaltous chloride:
Cobaltous chloride 2.0g
d. water  100 ml

Ammonium sulfide:
Ammonium sulfide 1.0 ml
tap water 10 ml  (but I was taught to use 20 ml) I don't know why. Does this need to be made fresh each time? Should the Ammonium sulfide be stored in the dark?

Incubation Solution:
Sodium barbital buffer 10.0 ml
Adenosine-5-triphosphate (ATP) 15mg
This also needs a pH of 9.4 and is prepared fresh each time. Again no pH modifications are done.

Basically when I do this stain it never looks the way it looks in the textbook. It always looks mottled. It never looks crisp with a distinct color difference.

Does this stain react differently with different muscles? If the muscle sample is of poor quality does it affect the staining?

I am also having trouble with the Modified Gomori Trichrome:

how important is it to stain the slides within one hour of cutting?  - I have not found it important 

does anyone filter the trichrome solution before staining?  - yes at least once/week 

I find this stain very basic yet it never comes out consistently. As of late, there is a dark , black  precipitate like artifact on the slides after staining. I'm not sure whats causing it.  - probably Haematoxylin ppt. Try filtering the Haematoxylin 

Heres the protocol:

Trichrome Solution
Fast Green FCF  0.03g
Chromotrope 2R 0.6g
Phosphotungstic Acid 0.6g
glacial acetic acid 1 ml
d. water 100 ml
Mix well and adjust pH to 3.4 ( again no pH adjusting here)

Stain slides in Harris Hematoxylin for 5 minutes.
Rinse in Distilled water  to remove excess Hemo.
Stain in trichrome solution for 20 minutes.
wash in distilled water to remove excess trichrome.
Dip twice in 0.2% acetic acid. ( we sometimes use 3%).
Dehydrate in graded alcohols and clear in xylene. Mount in synthetic medium.

I'll get back to everyone on the Nadh, Pas, Pasd, oro later. I don't want to bombard everyone. Feel free to post these on the histonet if its easier for you. Thanks to everyone who responded. I appreciate it.

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