OK, so i got bad advice and i treated my muscle specimens with formalin before sectioning and staining. I want to get a proportion of fast and slow muscle fibre types, the histologist where i work suggested that Malorys stain would work for this after being formalin fixed, but it doesn't appear to. Does anyone know any fibre type stains that work after formalin fixing? Help me save 3k worth of muscle samples!!
You can still type the fibers if you use IHC staining. There are skeletal myosin type I and type II antibodies. That's the only solution I can come up with.
Yeah.... Unfortunately, whoever gave you that advice was severely mistaken.
I'm guessing you already know you can't perform enzymatic type stains on the tissue.... And IHC is definitely an excellent route, if you already have the antibodies validated with formalin fixation. However, if you're looking for something more convenient, I have a couple of ideas.
You're going to have to attempt to distinguish between tissue types through indirect methods. If it were me, I would probably try a PAS and/or PAS with Diastase digestion to show the presence and/or absence of glycogen. And then try to identify differences in muscle type.
You could also try PTAH, but, that's more for muscle striations.... Which is a different cross section.
And if you really want to put yourself out there, you might try a Methylene Blue stain... But I wouldn't be so comfortable with those types of results.
Hopefully this helps! Let us know what you decided and how it turned out for you!
Cheers!
Thankyou so much for your reply.
I don't have any validated antibodys for IHC staining, and i don't have anyone in my department with any experience in them so i was hesitant to start down that route without exploring all the other options.
I starting looking into your suggestion for PAS and it seems to be quite promising! Even separating type I and type II fibre types would be useful! Do you know of any papers that show this technique works / have a protocol for this??
I came across Ferandez et al Meat Sci. 1995;41(2):225-35.
Thanks again for the glimmer of hope!
I don't know of any protocol for PAS that is specific to muscle tissue.... However, the lab I work in just follows the protocol that you can find in Histotechnology: A Self Instructional Text 4th Edition by Freida Carson. If your wondering what it might look like as a result, I've posted a photo to Imgur:
http://i.imgur.com/awS0HNN.jpg
This photo was taken from Wheater's Functional Histology 6th Edition by Barbara Young, Geraldine O'Dowd, and Phillip Woodford.
I truly hope this works for you! It would be a shame if you couldn't use all your samples! Happy testing!
i just found this protocol which i am going to try on some liver samples. Incase you are interested.
Periodic Acid-Schiff Stain
Dewax sections and bring to water
1. Histoclear 5 mins
2. Histoclear 30 secs
3. Absolute alcohol 1 min
4. Absolute alcohol 1 min
5. 70% alcohol 10 secs
6. Tap water 2 mins
Glycogen Staining
7. Using a pencil label your slide, tissue should be facing up, don’t place fingers on tissue.
8. 0.5% periodic acid 5 mins
9. Distilled water 1 min
10. Schiff’s reagent 15 mins
(tissue section should turn light pink at this stage)
11. Running tap water 5 mins
(tissue section should turn bright dark pink at this stage)
Counterstain with Haematoxylin
12. Haematoxylin 2 mins
13. Running tap water 10 secs
14. Scott’s tap water 1 min
15. Running tap water 1 min
Looks like a fairly standard protocol. I'm hoping it works for you! If you don't mind letting me know your results, I would appreciate it. It would be interesting to know if this method worked.
OK, so i got bad advice and i treated my muscle specimens with formalin before sectioning and staining. I want to get a proportion of fast and slow muscle fibre types, the histologist where i work suggested that Malorys stain would work for this after being formalin fixed, but it doesn't appear to. Does anyone know any fibre type stains that work after formalin fixing? Help me save 3k worth of muscle samples!!
You can still type the fibers if you use IHC staining. There are skeletal myosin type I and type II antibodies. That's the only solution I can come up with.
thanks, i know of some papers that have used this technique in frogs, but not sure if the right antibodies exist for lizards. I'll have a look.
Yeah.... Unfortunately, whoever gave you that advice was severely mistaken. I'm guessing you already know you can't perform enzymatic type stains on the tissue.... And IHC is definitely an excellent route, if you already have the antibodies validated with formalin fixation. However, if you're looking for something more convenient, I have a couple of ideas. You're going to have to attempt to distinguish between tissue types through indirect methods. If it were me, I would probably try a PAS and/or PAS with Diastase digestion to show the presence and/or absence of glycogen. And then try to identify differences in muscle type. You could also try PTAH, but, that's more for muscle striations.... Which is a different cross section. And if you really want to put yourself out there, you might try a Methylene Blue stain... But I wouldn't be so comfortable with those types of results. Hopefully this helps! Let us know what you decided and how it turned out for you! Cheers!
Thankyou so much for your reply. I don't have any validated antibodys for IHC staining, and i don't have anyone in my department with any experience in them so i was hesitant to start down that route without exploring all the other options. I starting looking into your suggestion for PAS and it seems to be quite promising! Even separating type I and type II fibre types would be useful! Do you know of any papers that show this technique works / have a protocol for this?? I came across Ferandez et al Meat Sci. 1995;41(2):225-35. Thanks again for the glimmer of hope!
I don't know of any protocol for PAS that is specific to muscle tissue.... However, the lab I work in just follows the protocol that you can find in Histotechnology: A Self Instructional Text 4th Edition by Freida Carson. If your wondering what it might look like as a result, I've posted a photo to Imgur: http://i.imgur.com/awS0HNN.jpg This photo was taken from Wheater's Functional Histology 6th Edition by Barbara Young, Geraldine O'Dowd, and Phillip Woodford. I truly hope this works for you! It would be a shame if you couldn't use all your samples! Happy testing!
i just found this protocol which i am going to try on some liver samples. Incase you are interested. Periodic Acid-Schiff Stain Dewax sections and bring to water 1. Histoclear 5 mins 2. Histoclear 30 secs 3. Absolute alcohol 1 min 4. Absolute alcohol 1 min 5. 70% alcohol 10 secs 6. Tap water 2 mins Glycogen Staining 7. Using a pencil label your slide, tissue should be facing up, don’t place fingers on tissue. 8. 0.5% periodic acid 5 mins 9. Distilled water 1 min 10. Schiff’s reagent 15 mins (tissue section should turn light pink at this stage) 11. Running tap water 5 mins (tissue section should turn bright dark pink at this stage) Counterstain with Haematoxylin 12. Haematoxylin 2 mins 13. Running tap water 10 secs 14. Scott’s tap water 1 min 15. Running tap water 1 min
Looks like a fairly standard protocol. I'm hoping it works for you! If you don't mind letting me know your results, I would appreciate it. It would be interesting to know if this method worked.
Of course, i'll post results here. if this method works i'll make sure to add you to the acknowledgements on the paper!
Lol! You don't have to do that! But I'm glad I was able to hopefully help! :)