- Catalog order # K7402 - $168.00 with Xylene
- K7604 - with xylene substitute $168.00
Rapid Frozen Section Biopsy Stain Kit Procedure for Dermatopathology
APPLICATION
Mammalian Tissue
DESIGNED TO SHOW
Excellent differential stain designed to show practically all cells. Cellular substances will stain with different shades.
SPECIMEN REQUIRMENTS
Unfixed Skin Tissue: Needs to be frozen immediately. To facilitate cutting and handling, apply an OCT compound to tissue before cutting. Use CO2, Nitrogen or Freeze spray to rapidly freeze specimen and OCT before using extractor.
Fixed Skin Tissue: Yields less damage under slower freezing conditions. Use Freon Aerosol or Freeze Spray will not effect tissue presentation. Rinse tissue thoroughly after fixation and blot to remove excess water before applying OCT.
Microscope Slides: For Dermatology biopsies it is recommended to use Poly-L-Lysine coated slides or Aminopropyltriethoxysilane (APES). For best results use positive charged slides. Slides available from IMEB, Inc.
EQUIPMENT
Cryostat temperature is important for cutting frozen sections. For skin biopsies keep temperatures around -15 to -18 C for unfixed tissue. For fixed tissues, keep temperatures around -8 to -15 C. As a rule, maintain cryostat temperatures at -20 C when not in use.
COMPONENTS AND REAGENTS
AF-110
32oz
32oz
32oz
32oz
1gal
1gal
1gal
1gal
1gal
16oz
Coverslips and two pair of forceps.
1 pair of Nitrile gloves (All sizes available)
(LAST FOUR ITEMS ARE NOT INCLUDED IN KIT)
PROCEDURE
Before beginning procedure, remove Station W from rack and place in sink.
1. Insert frozen sections in Station 1 for 60 seconds.
2. Rinse sections in Station W for 60 seconds.
3. Stain sections in Station 2 for 30 sections.
4. Rinse sections for 30 seconds in Station W.
5. Differentiate sections by inserting slides 1-2 times (1 second each) in Station 3.
6. Immediately transfer sections directly into Station 4 and treat for 60 seconds.
7. Rinse sections for 30 seconds in Station W.
8. Stain sections in Station 5 for 30 seconds with slow agitation.
9. Treat sections in Station 6 for 20 seconds.
10. Treat sections in Station 7 for 20 seconds.
11. Treat sections in Station A1 for 30 seconds.
12. Treat sections in Station A2 for 30 seconds.
13. Treat sections in Station A3 for 1 minute with slow agitation.
14. Clear sections in Station X for 1-2 minutes.
15. Coverslip sections with Mountant.
RESULTS
Nuclei - Blue
Cytoplasm - Pink
Fibrous tissue - Lilac
Elastica- Bright - Pink
Necrotic tissue - Shades of pink-purple
NOTES
1. Always check stock solutions against Station identification before pouring stock reageants.
2. Frozen sections should be immediately placed in fixative before or as soon as medium surrounding tissue begins to thaw.
3. "Fixed" frozen sections can be stored in an airtight container in the refrigerator at temperature around -40 until ready for use.
4. Forceps A should be used for Stations 1-W. Forceps B should be used for Stations 6-X.
SOLUTION MAINTENANCE
The following protocol is provided for daily use of kit. You may deviate from the following protocol pending on usage of kit.
1. Change Station 1 reagent biweekly.
2. Change Station W after each use.
3. Station 2 reagent may require weekly filtration. Change after two weeks. Always keep station sealed when not in use.
4. Change Station 4 reagent on a daily basis.
5. Change Station 5 reagent after two weeks. Keep station sealed to decrease evaporation of liquid.
6. Change Solutions 6-X on a biweekly basis.
TROUBLESHOOTING
1. Nuclear Staining too light: Increase staining time Station 2 or bypass Station 3 and transfer slides directly from Station 2 to Station 4.
2. Nuclear Staining too dark: Increase treatment time in Station 3 by an additional 1-2 seconds.
3. Cytoplasm Staining too light: Increase staining time in Station 5 by 30 seconds, reduce treatment in Station 6, 7, A1, and A2 by
10 seconds in each station. Dehydration through Station 6 – A2 is used to remove water, but also differentiate cytoplasm and background staining. Increasing or decreasing times in dehydrating Stations 6 – A2 will change the intensity of the Eosin Staining from Station 5.
4. Sections should not be left in Station X for more than two minutes to reduce possibility of removal of tissue section and stain.
5. Never contaminate Station A3 and X with water. This will cause bubbles, fogging and removal of stain from section.
6. IMEB has incorporated a copy of the H&E troubleshooting guide copied from the National Society of Histotechnology. This is a
helpful source of information provided for first time staining by lab technicians.
REFERENCES
1. Bancroft, J.D. (1975): Histochemical Techniques, 2nd Edition, pp. 20-46; Butterworths Inc., Boston.
2. Pearse, A.G.E. (1980): Histochemistry: Theoretical and Applied, 4th Edition, Vol. 1, pp. 22-27; Churchill Livingston, N.Y.
3. Sheehan, Denza and Hrapchak, B.: Theory and Practice of Histotechnology, 2nd Edition, St. Louis, Battelle Press, 1980.
4. Humason, G.L. (1972): Animal Tissue Techniques, 3rd Edition, pp. 148-149; W.H. Freeman & Co., San Francisco.
For further technical assistance, please call IMEB, Inc., phone:
1-800-543-8496: Mon.-Fri.: 8:00am-5:00pm.
