Hello,
I'm currently trying to stain 3D-micromass cell cultures obtained from differentiating equine mesenchymal stem cells in chondrogenic induction medium with Alcian Blue 8GX (done in 15 mL tubes).
For this i'm using the following protocol:
1. Make cryosections (8 µm sections) from 3D-micromass cultures (with OCT).
2. 8 µm sections were captures on SuperFrostPlus microscopic slides.
3. Fixation step for 10 min at RT with 4% PFA.
4. Wash three times in PBS buffer, for each wash dip the slides 10 times in 250 ml buffer.
5. Stain with Alcian Blue pH 2.5 solution for 30 min at room temperature in dark
6. Wash the slides in running tap water for 10 min, rinse in dH2O.
7. Examine.
However, i've tested different fixation times, different method of staining (in 4-well plate, without sectioning), however it seems like the staining is not working. It gives blue debris around the section. The staining solution was already made a couple of years ago (alcian blue 8GX + ethanol + glacial acetic acid), could this be the problem?
Thank you for your answers.
Hi Emma,
Seems to me that it could be a dye solubility or pH critical issue. Firstly, may I confirm what you are wishing to demonstrate? In purely aqueous solutions, Alcian Blues react with compounds containing anionic groups such as acid mucosubstances (MS), acid mucins and DNA. You have a tinge of alcohol in yours for reasons unknown. pH 2.5 will stain both sulphated and carboxylated (MS) pH 1.0 strictly sulphated (has different protocol). I would suggest you try a new fresh solution. Your OCT also should completely washed out before staining, but should come out in your pre-rinses.
0.1% Alcian Blue 8GX pH 2.5
Alcian Blue 8GX (BSC higher grade) 0.1 g
Glacial Acetic Acid 3 ml
Distilled water 97 ml
Check your final pH and filter paper before use.
specificity of staining will decrease, as alkalinity increases
Nuclear Fast Red Counterstain
Nuclear Fast Red 0.1g
Aluminum Sulphate 5g
Distilled Water 100ml
Thymol crystal 1 crystal
Heat water to 60C. Add aluminum sulphate. Stir until dissolved. Add Nuclear Fast Red. Cool to 50C and filter. Add Thymol. Keep solution refrigerated.
Staining Procedure:
1. Hydrate sections
2. Rinse in distilled water changes 2 minutes
3. Stain in Alcian Blue 30 minutes
4. Rinse in distilled water changes 3 minutes
5. Counterstain in Nuclear Fast Red 4 minutes
6. Rinse in distilled water
7. Dehydrate, clear, and mount.
Small intestine or cartilage serves as a nice positive control.
Good luck, Emma!
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