Congo Red

Author: Kelsey Goldman, HTL (ASCP)

Last Update: 16 July 2023

Copyright: 2023, HistologyOutlines.com

Cite this Page! -> Goldman, Kelsey. “Congo Red.” HistologyOutlines.Com, 16 July 2023. Accessed (today) 

Special Stain

Purpose:
  • To demonstrate the presence/ absence of Amyloid in a tissue.
    Control Tissues:
  • FFPE section of known Amyloidosis. Must be sectioned within 8-10 microns. May labs prefer the control and experimental/patient set of slides to be fresh cut (ie sectioned within 24 hours of staining).
    Staining Pattern:
  • Viewed in a polarized microscope- classic “apple-green birefringence” in areas with amyloid
  • Fluorescent microscope- positive staining causes a spectral shift from 490mm to 515mm
  • Bright-field Microscope- areas of red/ orange staining in areas of amyloidosis
    Biochemistry Theory:
  • The biochemical properties that lead to positive staining in Congo Red are poorly understood. It is thought that a properly made, appropriately linear molecule of Congo Red has affinity for the beta pleated sheets that are the common make up of amyloid.
    Images:
    Artifacts: 
  • Heat from surgical cautery may cause fringe false positive staining
  • In addition to amyloid Congo Red will also stain elastin and collagen
  • Sections cut less than 5 microns may exhibit false positive staining. It is important to align the thickness with the labs protocol and it is typically recommended to remain between 8-10 microns.


    Sample Protocol:
    • -Prepare the following stock solutions in advance of staining:
    • 0.5% Congo Red in 50% Alcohol

    • 0.2% Potassium Hydroxide in 80% Alcohol


      • -Hydrate Sections to Water

      -Stain in a 0.5% Congo Red Solution in 50% Alcohol for 5 minutes

      -Differentiate in Alcoholic  Potassium Hydroxide solution for 3-10 seconds. DO NOT over differentiate. Dip slides into water to stop the differentiation  process. Check on microscope to verify appropriate staining. The side is over differentiated if you do not see any areas of deep red in your control. If this occurs place the slide back into the prepared Congo Red step  for an  5 minutes and repeat differentiation.

      -Wash in deionized water

      -Stain with an Alum Hematoxylin, differentiate and blue as appropriate

      -Dehydrate, clear and mount

      Adapted  from Suvarna, S. Kim, et al., editors. Bancroft’s Theory and Practice of Histological Techniques. Eighth edition, Elsevier, 2019.

    – Prepare the following:

  • Dissolve 0.5g of potassium hydroxide in 50 mL of distilled water. Add 200 mL of 100% alcohol to this solution. Add Congo Red to this solution until it is saturated with the Congo Red. This final solution needs to sit overnight and can be kept for 3 months

    • -Hydrate sections

    -Stain in filtered Congo Red solution for 25 minutes

    -Dip in deionized water

    -Differentiate in running tap water for 5 minutes

    -Counterstain in alum hematoxylin as appropriate. differentiate and blue as necessary

    -Dehydrate, clear and mount

    Adapted from Suvarna, S. Kim, et al., editors. Bancroft’s Theory and Practice of Histological Techniques. Eighth edition, Elsevier, 2019.
    -Prepare the following stock solutions in advance of staining:
  • Solution A- Saturated Sodium Chloride in 80% Ethanol
  • Solution B- Saturated Congo Red in 80% Ethanol with Sodium Chloride. Allow Solution B to sit overnight before use
  • 1% Aqueous Sodium Hydroxide

    • -Prepare the following working solutions less than 20 minutes before staining:
  • Working Solution A-Take 100ml of the previously made Solution A and add 1mL of the 1% Sodium Hydroxide. Filter.
  • Working Solution B-Take 100 mL of the previously made Solution B and add 1mL of the 1% Sodium Hydroxide. Filter


    • -Hydrate the sections to water

    -Stain and Blue with an Alum Hematoxylin

    -Place sections into working solution A for 20 minutes

    – Drain, do not wash or blot dry the sections

    -Place sections into working solution B for 20 minutes

    – Rinse quickly in alcohol, clear in xylenes and coverslip

    Adapted from Suvarna, S. Kim, et al., editors. Bancroft’s Theory and Practice of Histological Techniques. Eighth edition, Elsevier, 2019.
    Additional References:
  • Rambaran, Roma N., and Louise C. Serpell. “Amyloid Fibrils.” Prion, vol. 2, no. 3, 2008, pp. 112–17, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634529/.
  • Suvarna, S. Kim, et al., editors. Bancroft’s Theory and Practice of Histological Techniques. Eighth edition, Elsevier, 2019.
  • Yakupova, Elmira I., et al. “Congo Red and Amyloids: History and Relationship.” Bioscience Reports, vol. 39, no. 1, Jan. 2019, p. BSR20181415, https://doi.org/10.1042/BSR20181415.