Calcium
Validation Stain: Von Kossa
Other Applicable Stains: Alizarin Red
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PRODUCT SPECIFICATIONS:
Tissue: Positive staining placenta.
Fixation: Formalin 10%, Phosphate Buffered (Part 1090).
Section/Glass: Paraffin sections cut at 4 microns on Superfrost™ Plus slides.
Quality Control Stain: Von Kossa Calcium quality control stained slide(s) included.
Reactivity: Guaranteed product specific reactivity for one year from date of receipt. Revalidate after one year to verify continued reactivity.
Storage: 15-30°C in a light deprived and humidity controlled environment.
Intended Use: To verify histological techniques and reagent reactivity.
Before using unstained control slides, review the enclosed stained slide(s) to ensure that this tissue source is acceptable for testing needs.
CONTROL SLIDE VALIDATION:
With Von Kossa Calcium Stain: | Individual Stain Solution |
Silver Nitrate 5%, Aqueous | Part 13805 |
Sodium Thiosulfate 5%, Aqueous | Part 1389 |
Nuclear Fast Red Stain, Kernechtrot | Part 1255 |
APPLICATION:
Newcomer Supply Calcium Control Slides are for the positive histochemical staining of calcium or calcium salts in tissue sections.
PRESTAINING PREPARATION:
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- Heat dry sections in oven according to you laboratory protocol.
- All glassware/plasticware must be acid cleaned prior to use.
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- See Procedure Notes #1 and #2
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NEWCOMER SUPPLY VALIDATION PROCEDURE:
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- 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.
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- See Procedure Notes #3 and #4.
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- Place slides in Silver Nitrate 5%, Aqueous according to the following timings and conditions.
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- Direct sunlight or ultraviolet light for 10-30 minutes.
- In front of a 60-100 watt light bulb for 1 hour or longer.
- See Procedure Note #5.
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- Check slides periodically and remove from light source when control slide shows black-brown deposits macroscopically.
- Rinse well in several changes of distilled water.
- Place slides in Sodium Thiosulfate 5%, Aqueous for 2 minutes.
- Rinse well in several changes of distilled water.
- Counterstain in Nuclear Fast Red Stain, Kernechtrot for 5 minutes.
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- Shake solution well before use; do not filter.
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- Rinse well in distilled water.
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- See Procedure Note #6.
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- 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.
- 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.
RESULTS:
Calcium salts | Black to brown/black |
Nuclei | Red |
Cytoplasm | Light pink |
PROCEDURE NOTES:
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- Acid clean all glassware/plasticware (Part 12086) and rinse thoroughly in several changes of distilled water.
- No metals of any kind should come in contact with silver solutions to prevent precipitation of silver salts. Use plastic forceps (5500) or paraffin coated metal forceps.
- Drain slides after each step to prevent solution carry over.
- Do not allow sections to dry out at any point during procedure.
- Direct sunlight is the preferred method. If procedure is in minimal sunlight increased incubation time will be necessary.
- Wash well after Nuclear Fast Red Stain, Kernechtrot to avoid cloudiness in dehydration steps.
- If using a xylene substitute, follow manufacturer’s recommendation for deparaffinization and clearing steps.
REFERENCES:
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- Carson, Freida L. and Christa Hladik. Histotechnology: A Self-Instructional Text. 3rd ed. Chicago, Ill.: American Society of Clinical Pathologists, 2009. 269-270.
- Sheehan, Dezna C. and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 226-227.
- Modifications developed by Newcomer Supply Laboratory.