Alizarin Red S Stain Protocol for Calcium

Run a control slide. Control tissue: Bone or tissue with calcium present

Safety equipment: Work under a hood with lab coat, gloves, and safety glasses.

  1.   De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
  2.   De-paraffin slides in xylene (2)——————- 2 minutes (re-use)
  3.   Clear slides in 100% alcohol———————- 2 minutes
  4.   Clear slides in 100% alcohol———————- 2 minutes
  5.   Hydrate slides in 95% alcohol——————– 2 minutes
  6.   Hydrate slides in 50% alcohol——————– 1 minute
  7.   Rinse rapidly in DH2O—————————— 30 seconds
  8.   Cover the sections with alizarin Red S solution (re-use)
  9.   Observe the reaction under a microscope and remove when the red-orange lake forms ( 5 minutes to 15 mintues). The lake should be heavy but not too diffuse.

10.    Shake off excess dye and blot carefully.

11.    Dehydrate in acetone for 10 to 20 seconds (dump)

12.    Dehydrate in acetone-xylene (50:50)———–10 to 20 seconds (dump)

13.    Clear in xylene—————————————- 5 minutes (re-use)

14.    Coverslip

Results:

Calcium Deposits (except oxalate) ———————Orange-Red

This precipitate is birefringent.

 

An Expert Histology Service


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