Von Kossa Calcium Stain

Reagents for this procedure are sold as individual stain solutions and are available for purchase under separate part numbers with storage requirements and expiration date designated per bottle.

Product Options:

Part # Non-Kit

SOLUTIONS: 250 ml 500 ml 1 Liter
Silver Nitrate 5%, Aqueous Part 13805A Part 13805B
Sodium Thiosulfate 5%, Aqueous Part 1389A Part 1389B
Nuclear Fast Red Stain, Kernechtrot Part 1255A Part 1255C Part 1255B

 

Additionally Needed:

Calcium Control Slides Part 4100
Acid 5%, Aqueous Part 12086 (for acid cleaning glassware)
Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842

For storage requirements and expiration date refer to individual product labels.

 

APPLICATION:

Newcomer Supply Von Kossa Calcium Stain is an indirect staining method for the demonstration of calcium or calcium salt in tissue sections. This technique is not specific for calcium. Other reducing substances, such as formalin pigment and melanin, will also give a positive reaction.

 

METHOD:

Fixation: Alcohol or Formalin 10%, Phosphate Buffered (Part 1090)

Technique:  Paraffin sections cut at 4 microns

Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

All Newcomer Supply stain procedures are designed to be used with Coplin jars filled to 40 ml following the provided staining procedure.

 

PRESTAINING PREPARATION:

    1. If necessary, heat dry tissue sections/slides in oven.
    2. All glassware/plasticware must be acid cleaned prior to use.
        1. See Procedure Notes #1 and #2.

 

STAINING PROCEDURE:

    1. Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each. Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each.  Wash well with distilled water.
        1. See Procedure Notes #3 and #4.
    2. Place in Silver Nitrate 5%, Aqueous (Part 13805) according to the following timings and conditions.
        1. Direct sunlight or ultraviolet light for 10-30 minutes.
        2. In front of a 60-100 watt light bulb for 1 hour or longer.
        3. See Procedure Note #5.
    3. Check slides periodically and remove from light source when control slide shows black-brown deposits macroscopically.
    4. Rinse well in several changes of distilled water.
    5. Place in Sodium Thiosulfate 5%, Aqueous (Part 1389) for 2 minutes.
    6. Rinse well in several changes of distilled water.
    7. Counterstain in Nuclear Fast Red Stain, Kernechtrot (Part 1255) for 5 minutes.
        1. Shake solution well before use; do not filter.    
    8. Rinse well in distilled water.
        1. See Procedure Note #6.
    9. Dehydrate in two changes each of 95% and 100% ethyl alcohol; 10 dips each. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

 

RESULTS:

Calcium salts Black to brown/black
Nuclei Red
Cytoplasm Light pink

 

PROCEDURE NOTES:

    1. Acid clean all glassware/plasticware (Part 12086) and rinse thoroughly in several changes of distilled water.
    2. Plastic (Part 5500), plastic-tipped, or paraffin coated metal forceps must be used with any silver solution to prevent precipitation of silver salts. No metals of any kind should be in contact with any silver solution.
    3. Drain slides after each step to prevent solution carry over.
    4. Do not allow sections to dry out at any point during procedure.
    5. Direct sunlight is the preferred method. If procedure is carried out in minimal sunlight increased incubation time will be necessary.
    6. Wash well after Nuclear Fast Red Stain, Kernechtrot to avoid cloudiness in dehydration steps.
    7. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

 

REFERENCES:

    1. Carson, Freida L., and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 269-270.
    2. Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 226-227.
    3. Modifications developed by Newcomer Supply Laboratory.