VitroView Safranin O Stain Kit for Cartilage (#VB-3028)
 
Description
Safranin O is a cationic dye which binds specifically to polyanions. It detects the amount of proteoglycan present in cartilages. The intensity of Safranin O staining is proportional to the proteoglycan content in the cartilage tissue. It is used to detect any variations in articular disease as a marker for glycosaminoglycan depletion in osteoarthritis. Fast Green counterstains the non-collagen sites and provides a clear contrast to the Safranin O staining.
This VitroView™ Safranin O Stain kit is used for the detection of cartilage, mucin, and mast cell granules on formalin-fixed, paraffin-embedded tissue sections, and may be used for frozen sections as well. The cartilage and mucin will be stained orange to red, and the nuclei will be stained black. The background is stained bluish green
 
Kit Components
SKU#
Reagent Name
Volume (ml)
VB-3028-1
Weigert’s Hematoxylin Solution A
125
VB-3028-2
Weigert’s Hematoxylin Solution B
125
VB-3028-3
Acid Ethanol
250
VB-3028-4
Fast Green (FCF) Solution
250
VB-3028-5
Acetic Acid Solution
250
VB-3028-6
Safranin O Solution
250
 
Storage
Room temperature.
 
Protocol
Safranin O Stain for paraffin sections
Decalcified paraffin sections cut at 4 microns
Deparaffinize in xylene I for 6 minutes and II for 6 minutes.
 Rehydrate
Ethanol 100% (2 minutes)
Ethanol 100% (2 minutes)
Ethanol 95% (2 minutes)
Ethanol 95% (2 minutes)
Ethanol 70% (2 minutes)
Rinse in DI water (2 minutes).
Prepare Weigert’s working hematoxylin: mix Weigert’s Hematoxylin Solution A and B at 1:1 ratio.
Weigert’s working hematoxylin for 10 minutes (Note: Discard after use).
Blue in running tap water for 5 minutes.
Destain quickly in Acid ethanol (2-3 dips).
Wash in tap water for 2 minutes, rinse once for 1 min in distilled water.
Stain with fast green (FCF) solution for 5 minutes.
 Rinse quickly with acetic acid solution for 2-3 dips.
Stain in safranin O solution for 5 minutes.
Dehydrate with 2 changes of 100% Ethanol (2 minute per change).
Clear with 2 changes of xylene (5 minute per change).
Mount cover slip onto glass slide with Permount or some other suitable organic mounting medium.
 
Safranin O Stain  Stain for frozen sections
Frozen section are fixed in acetone at -20 °C for 15 min or 80% Methanol at 4°C for 5 min, or 10% neutralized formalin for 10 min.
PBS 1× 10 min.
Rinse in DI water (2 minutes).
Follow step 4-15 above.
 
Expected results
Nuclei—————————————————Black
Cytoplasm——————————————-Gray green
Cartilage, mucin, mast cell granules———–Orange to red
 
Control Tissue:
Cartilage