Luxol Fast Blue (LFB) Stain Set

Product Options:

Part # 12218A 500 ml set $73.00 H
Part # 12218B 1 L set $104.00 H

   

LUXOL FAST BLUE (LFB) STAIN SET INCLUDES:                                                                                               

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.


APPLICATION:

Newcomer Supply Luxol Fast Blue (LFB) Stain Set, with included microwave modification, is a common procedure for the demonstration of myelin in central nervous system and peripheral nerve tissues.

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


METHOD:
 

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 provided staining procedure.  Some solutions in the set may contain extra volumes.


PRESTAINING PREPARATION:

    1. If necessary, heat dry tissue sections/slides in oven.
    2. Prepare Working Lithium Carbonate 0.05%; combine and mix well;
      1. Solution B: Lithium Carbonate, Saturated Aqueous 5 ml
      2. Distilled Water                     95 ml


LFB/H&E 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.
      1. Stop at 95% ethyl alcohol; no distilled water rinse.
      2. See Procedure Notes #1 and #2.
    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.
      1. To enhance stain, 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.

      1. Place slides in a plastic Coplin jar with Solution A: Luxol Fast Blue Stain 0.1%, Alcoholic. Microwave at 70°C for 10 minutes.
    1. Rinse slides quickly in 95% ethyl alcohol (Part 10842); 2-3 dips.
    2. Rinse in distilled water.
    3. Differentiate slides individually in Working Lithium Carbonate 0.05% (Step #2) for 10-15 seconds with agitation until gray and white matter are colorless and contrast with stained tissue.
    4. Further differentiate in 70% ethyl alcohol (Part 10844), until gray and white matter can be distinguished. Do not over differentiate.
    5. Rinse in distilled water.
    6. Check slides microscopically. Continue if additional differentiation is needed. Otherwise proceed directly to Step #12.
      1. One dip in Lithium Carbonate 0.05%, Aqueous (Step #2).
      2. Dip in two changes of 70% ethyl alcohol until green/blue white matter sharply contrasts with colorless gray matter.
    1. Rinse thoroughly in distilled water.
    2. Stain with Hematoxylin Stain, Harris Modified (Part 1201) for 1-5 minutes, depending on preference of intensity.
    3. Wash in running tap water for 3 minutes.
    4. Differentiate quickly in Acid Alcohol 1% (Part 10011); 3 dips.
    5. Wash well in running tap water.
    6. Blue in Solution B: Lithium Carbonate, Saturated Aqueous.
    7. Wash well in running tap water.
    8. Counterstain in Eosin Y Working Solution (Part 1072) for 30 seconds to 3 minutes, depending on preference of intensity.
    9. 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.


RESULTS:

Myelin (white matter) Blue to blue/green
Gray matter and cytoplasm Shades of pink to red
Nuclei Dark blue

 

PROCEDURE NOTES:

    1. Drain slides after each step to prevent solution carry over.
    2. Do not allow sections to dry out at any point during procedure.
    3. 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.
    4. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.


REFERENCES:

    1. Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press, 2015. 206-211.
    2. Klüver, Heinrich, and Elizabeth Barrera. “A Method for the Combined Staining of Cells and Fibers in the Nervous System.” Journal of Neuropathology and Experimental Neurology4 (1953): 400-403.
    3. Luna, Lee G. Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. Gaitheresburg, MD: American Histolabs, 1992. 494-495.
    4. Modifications developed by Newcomer Supply Laboratory.