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Desmin - Science topic

An intermediate filament protein found predominantly in smooth, skeletal, and cardiac muscle cells. Localized at the Z line. MW 50,000 to 55,000 is species dependent.
Questions related to Desmin
Şeyda Ünsal
Şeyda Ünsal
  • asked a question related to Desmin
How to get rid of immunfluorescent staining in negative control caused by secondary antibody?
Question
3 answers
  • 19 Oct 2018
I am currently tryig to do double immunfluorescent staining in myotubes but I could not be able to see a clear negative control.
As you may see in the presentation, I used two different kinds of negative control. 1) no primary antibody 2) Another cell line that should not be expressing desmin (fibroblast). In both of my negative controls I still have strong staining, especially localized in nuclei that I could not get rid of.
%4 PFA is my fixative agent. I am using donkey anti-rabbit (AF488) and donkey anti-goat (AF568) secondary antibodies in 1/5000 dilution.There is not any negative staining in Texas red but in FITC. I do all the washes with %0,5 PBS(T) and 15 minutes. I used 1% FBS and 5% BSA blocking but there was no change. Moreover, I changed the cell type and do the experiment in C2C12 myoblasts but still the same result. Finally, I did desmin staining on its own but negative control was not clear again with donkey anti-rabbit (AF488) but when we do double staining in skeletal muscle tissue, the result was satisfying; no staining observed at negative control.
What could have gone wrong with the cells or antibodies? Any ideas?
Thank you for your kind answers.
Relevant answer
  • 20 Oct 2018
Answer
Dear Seyda,
One problem may be autofluorescence from Paraformaldehyde, which is typically seen in the FITC (488) channel. If you take some cells run them through your protocol, but do not apply any antibodies at all (either primary or secondary), do you still see a light green signal?
If so, there are methods to quench autofluorescence. I usually use 0.1 M glycine in PBS, pH 7.4, as a 30 minute rinse, before adding my blocking agent, on 11um-20um tissue sections. If you don't have any glycine, lysine works too.
Otherwise, try using a different wavelength fluorochrome. Jackson ImmunoResearch has a large variety of Donkey anti-rabbit secondaries that are useful for double-labeling (note: no financial interest).
Good Luck!
Jill
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Xiaomei Yang
Xiaomei Yang
  • asked a question related to Desmin
What markers can I use to detect myofibroblasts?
Question
4 answers
  • 11 Sept 2017
I am trying to study if a special  molecular could induce fibroblast differentiate into myofibroblast. I have used a classic marker SMA and got a positive result in this study. But I need more markers to verify the result. Can I use desmin or SM22a as myofibroblast markers?
Welcome to discuss with me about this topic!
Thx
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  • 4 Sept 2018
Answer
I wish I had come across this post earlier. Were you able to select a marker besides alpha-SMA? Aren't myofibroblasts negative for desmin?
I know it has been long. But I am trying to quantify myofibroblasts too but post-infarction from heart tissue. Kindly share you experience and positive results. Help appreciated!
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Vishnu Vignesh Kanagaraj
Vishnu Vignesh Kanagaraj
  • asked a question related to Desmin
Could someone explain why there is upregulation of desmin and vimentin genes in xenobiotic treated skeletal muscle of rats ?
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3 answers
  • 1 Aug 2015
desmin and vimentin shows upregulation at gene level of skeletal muscle treated with fluoride. please provide me with possible reasons.
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  • 6 Aug 2015
Answer
We need know more detail about  your research design for your question.
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Ketaki Mhatre
Ketaki Mhatre
  • asked a question related to Desmin
Why, as I am optimizing Desmin staining of Neonatal Rat cardiomyocytes, I am getting weak staining with my Sigma Desmin Antibody?
Question
3 answers
  • 7 Jun 2015
So, I fix the cells with Acetone:Methanol and permeabilize the cells before staining.
Please do suggest where should I start optimizing my protocol.
Thanks!
Relevant answer
  • 8 Jun 2015
Answer
Hi Ketaki,
May be  weak staining indicating the low expression of Desmin after your treatment of cardiomyocytes.
Also sometimes methanol due to auto florescence subside antibody signal. You can increase concentration of primary antibody or try antibody from different company. Best of Luck.
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Hoda M. Eltaher
Hoda M. Eltaher
  • asked a question related to Desmin
How fast can myogenic markers be detected using immunohistochemistry?
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5 answers
  • 22 Apr 2015
Upon performing reprogramming/differentiation over 3 days experiment, can desmin, MyoD or alpha actinin be detected this early?
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  • 23 Apr 2015
Answer
For SMCs cultured on 3 D scaffolds, smooth muscle a-actin can be detected as early as 24 h (early marker), calponin at 3-4 days (middle marker), while Myosin heavy chain (late marker) would need more than 7-day culture to be expressed. 2D cultures/ TCPS might take shorter time.
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