Luxol Fast Blue (LFB) Stain Set
|Part 12218A||Part 12218B|
|Solution A:||Luxol Fast Blue Stain 0.1%, Alcoholic||500 ml||1000 ml|
|Solution B:||Lithium Carbonate, Saturated Aqueous||500 ml||1000 ml|
Additionally Needed For LFB/H&E Stain:
|Luxol Fast Blue (LFB) Control Slides||Part 4407|
|Hematoxylin Stain, Harris Modified||Part 1201|
|Acid Alcohol 1%||Part 10011|
|Eosin Y Working Solution||Part 1072|
|Xylene, ACS||Part 1445|
|Alcohol, Ethyl Denatured, 100%||Part 10841|
|Alcohol, Ethyl Denatured, 95%||Part 10842|
|Alcohol, Ethyl Denatured, 70%||Part 10844|
|Coplin Jar, Plastic||Part 5184 (for microwave modification)|
For storage requirements and expiration date refer to individual product labels.
Newcomer Supply Luxol Fast Blue (LFB) Stain Set, with included microwave modification, is a commonly used procedure for the demonstration of myelin in central nervous system tissues and in peripheral nerve.
The LFB Stain Set is flexible and can be used as a stand-alone without additional stain/counterstain or combined with options such as:
- LFB/Hematoxylin or LFB/Hematoxylin and Eosin (H&E)
- LFB/PAS or LFB/PAS/Hematoxylin
- LFB/Cresyl Violet
- LFB/Nuclear Fast Red
- LFB/Silver Nitrate
Staining results will vary according to option used.
Fixation: Formalin 10%, Phosphate Buffered (Part 1090)
Technique: Paraffin sections cut at 8-10 microns on adhesive slides
Solutions: All solutions are manufactured by Newcomer Supply, Inc.
All Newcomer Supply Stain Sets are designed to be used with Coplin jars filled to 40 ml following the staining procedure provided below. Some solutions in the set may contain extra volumes.
LFB/H&E STAINING PROCEDURE:
- 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.
- Stop at 95% ethyl alcohol; no distilled water rinse.
- See Procedure Notes #1 and #2.
- Incubate slides in Solution A: Luxol Fast Blue Stain 0.1%, Alcoholic for 2 hours at 60°C or overnight at 37°C; seal lids tightly.
- To enhance staining and differentiation process for both regular and microwave modification, add 0.4 ml of Acetic Acid, Glacial, ACS (Part 10010) to 40 ml of Solution A: Luxol Fast Blue Stain 0.1%, Alcoholic before use.
Microwave Modification: See Procedure Note #3.
- Place slides in a plastic Coplin jar containing Solution A: Luxol Fast Blue Stain 0.1%, Alcoholic and microwave at 70°C for 10 minutes.
- Rinse slides quickly in 95% ethyl alcohol (Part 10842); 2-3 dips.
- Rinse in distilled water.
- Prepare Working Lithium Carbonate 0.05%; combine and mix well.
- Solution B: Lithium Carbonate, Saturated Aqueous 2 ml
- Distilled Water 38 ml
- Differentiate each slide individually in Working Lithium Carbonate 0.05% for 10-20 seconds with agitation until gray matter and white matter are colorless and in high contrast with stained tissue.
- Save solution for reuse in Step #9a.
- Further differentiate in 70% ethyl alcohol (Part 10844), until gray and white matter can be distinguished. Do not over differentiate.
- Rinse distilled water.
- Complete differentiation:
- Rinse slides briefly in Working Lithium Carbonate 0.05%.
- Rinse in two changes of 70% ethyl alcohol until green/blue white matter contrasts with colorless gray matter.
- Rinse thoroughly in distilled water.
- Stain with Hematoxylin Stain, Harris Modified (Part 1201) for 1-5 minutes, depending on preference of stain intensity.
- Wash in running tap water for 3 minutes.
- Differentiate quickly in Acid Alcohol 1% (Part 10011); 3 dips.
- Wash well in running tap water.
- Blue slides in Solution B: Lithium Carbonate, Saturated Aqueous.
- Wash well in running tap water.
- Counterstain in Eosin Y Working Solution (Part 1072) for 30 seconds to 3 minutes, depending on preference of stain intensity.
- Dehydrate in two changes of 95% for 1 minute each and two changes of 100% ethyl alcohol, 10 dips each. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.
|Myelin (white matter)||Blue to blue/green|
|Gray matter and cytoplasm||Shades of pink to red|
- Drain slides after each step to prevent solution carry over.
- Do not allow sections to dry out at any point during procedure.
- The suggested microwave procedure has been tested at Newcomer Supply. This procedure is a guideline and techniques should be developed for use in your laboratory.
- If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.
- Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press, 2015. 206-211.
- Klüver, Heinrich, and Elizabeth Barrera. “A Method for the Combined Staining of Cells and Fibers in the Nervous System.” Journal of Neuropathology and Experimental Neurology 12.4 (1953): 400-403.
- Luna, Lee G. Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. Gaitheresburg, MD: American Histolabs, 1992. 494-495.
- Modifications developed by Newcomer Supply Laboratory