I stained embryonic chondrogenesis in-vitro by Alcain blue using following protocol, but the chondrocyte didn't stain.
1) Fix cultured cells with Paraformaldehyde 4% (W/V) for 20min for
2) rinse 3 times with 1x PBS
3) stain in alcian blue solution for overnight
4) rinse twice with 1x PBS
5) add Guanidin hydrochloride overnight
6) read the absorption on 600 nm
Hi Fereshteh,
What stage of embryonic chondrocyte you are culturing, are you taking any control cartilage from any animal like mouse, rat, human.
Second, are you adjusting pH of Alcian blue to 2.5 or so.
You may go to "Alcian Blue protocol at IHC World" on Google, you may get some hints if you are doing any mistake. You should take positive control, that will help you, or some chondrocytes from adults
Best of luck,
Subhash
Hi Subhash
thanks for your attention
I obtained embryonic cell from a rat at 13-day gestation age and made single cells by trypsin. the cells were seeds on 24-well plate and after 8 days of seeding, I did Alcian blue staining for chondrocyte staining.
the PH of Alcian blue solution was 1, is this the unsuccessful staining?
pH 2,5 stains carboxylated and sulfated proteoglycans,
pH 1 stains only sulfated proteoglycans. If the amount of those proteoglycans is very small, it may be too faint to see.
Take Alcianblue pH 2,5 and prolong staining until wished intensity.
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