Hello I am performing the chondrogenic differentiation of adipose-derived stem cells in bidimensional cultures.
I would like to ask which one is the best protocol for the staining and the semi-quantification by absorbance at 630 nm.
I read several protocols... I have Alcian Blue 8GX (powder) from sigma (A5268). I saw in short, two kind of staining with Alcian blue in Acetic acid (pH - 2.5) and Alcian blue in 0.1 M HCl overnight after 20 min of fixation in 95% methanol (also I have doubts with the fixation... formalin, PFA 4%...?). The second one seems to be the best for cell layers in 2D, however I am a little afraid of the 0.1 M HCl (Did anybody tried it?).
Finally for the extraction of Alcian blue in order to make the quantification I read 6h of incubation in Guanidine-HCl 6M, correct?
Thanks in advanced,
Sergio.
Hello
Yes for cell layers in 2D , i think the second one is the best
Good Luck
Second one. If you have your cells fixed prior to staining no problem. Also notice that the ph of the alcian blue may differ which GAG they will stain. pH 1.0 will stain sulphated mucins while ph2.5 will stain carboxylated mucins. Cheers!
We've had good luck with the following protocol on fixed samples in 6-well plates:
1) Rinse wells 3x with PBS.
2) Rinse 1x with 1% acetic acid. 3) Apply enough Alcian blue solution to cover wells. Incubate 15mins.
4) Rinse well repeatedly with PBS until clear. Dry plates in hood.
5) Measure absorbance at 630 nm
Kaitlin Fogg Can u plz give any reference for this method.Any published work?
Samiya Riaz - Of course, sorry I forgot to include it the first time!
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