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Specifically, I'd like to look at changes in HA (hyaluronic acid) production. Most often you only see people staining the cell surface or removing cells from culture for fixation and then imaging. Does anyone know how to analyze the ECM (extracellular matrix) and proteoglycans that surround a cell if you're looking to see if a condition may be changing the way a cell remodels it's ECM? Is it even possible with some fluorescent tags?

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    $\begingroup$ Can you define some of those acronyms? I'm assuming ECM is extracellular matrix, and HA is hyalauronic acid, but I could be wrong, and I could have spelled hyalauronic wrong. $\endgroup$ – user137 Oct 17 '14 at 22:49
  • $\begingroup$ Yes, sorry---ECM = extracellular matrix, HA = hyaluronic acid. I know that a typical hematoxylin and eosin stain (H & E) will pick up things that are negatively charged, such as HA, but how can you fix cells without disrupting the ECM? Also, I was wondering if methods exist to analyze cell ECM production via fluorescence or some other method that utilizes a probe that I could observe with a microscope. $\endgroup$ – GPI Oct 17 '14 at 23:13
  • $\begingroup$ Interesting question. Afaik ECM is much more than just HA, it contains chondoitin and other glucoseamine related stuff as well. Some of them is capable to regulate immune reaction, for example chondroitin-sulfate can reduce TNFa production and so inflammation by arthritis. $\endgroup$ – inf3rno Oct 18 '14 at 13:00
  • $\begingroup$ naturebiotech.com.tw/Fluorescent%20ECM%20Proteins.pdf I guess this study uses the same stuff: figshare.com/articles/… it is probably possible, but I never worked with ECM. $\endgroup$ – inf3rno Oct 18 '14 at 20:01
  • $\begingroup$ There have got to be some antibodies against ECM components you could use for immunohistochemical labeling. $\endgroup$ – user137 Oct 18 '14 at 22:15
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Different components of the ECM can be stained differently but since you asked about hyaluronic acid (HA) I'll limit my answer to its staining.

See this paper. They use Hyaluran Binding Protein (HABP) as a specific probe for HA.

Because HA has a very simple, conserved composition and is ubiquitously expressed in all animals that have a developed immune response, HA is not immunogenic. Therefore, there are no antibodies that specifically recognize HA, and traditional immunohistochemical methods of detection of HA are not possible. Fortunately, a very specific and tightly binding protein, the hyaluronan binding protein (HABP), which is composed of the HA binding domain with the link module from aggrecan, was isolated (Hascall and Heinegård 1974; Tengblad 1979) and adapted as an HA probe (Ripellino et al. 1985). HABP is now widely employed for specific detection of HA.

Biotin tagged HABP can be used for IHC based visualization procedures.

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The book "Basic Histology" by Junqueira and Carneiro acknowledge the problem in histologic fixation of ground substance. They recommend freeze drying in liquid nitrogen and then removing water by high vacuum at a temperature of -30 celcius, which will cause sublimation (ice-->vapor). Then fixing the sample with a nonpolar solvent. Afterwards you can use stains like PAS technic.

I've never done this, I'm just copying from the book. If you already happen to have liquid nitrogen, high vacuum, and PAS in your lab this might be cheaper than HABP.

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