Related Experiment Video
Updated: Aug 28, 2025
Author Spotlight: Effective Immunofluorescent Staining Protocol for Intestinal Cell Biology Research
Published on: July 19, 2024
Optimizing immunostaining of archival fish samples to enhance museum collection potential
Garfield T Kwan1, Benjamin W Frable2, Andrew R Thompson3
1Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, USA; NOAA Fisheries Service, Southwest Fisheries Science Center, La Jolla, CA, USA.
Insights
This study introduces a sodium borohydride (SBH) pretreatment to reduce autofluorescence in archival fish specimens. This method enables successful immunohistochemistry (IHC) on old samples, revealing biological responses to environmental changes.
Area of Science:
- Ichthyology
- Biochemistry
- Histology
Background:
- Immunohistochemistry (IHC) is vital for visualizing proteins in tissues.
- Autofluorescence in fluid-preserved natural history specimens significantly hinders IHC signal quality.
- Existing methods struggle with high background noise in archival samples.
Purpose of the Study:
- To evaluate autofluorescence reduction techniques for IHC on archival fish specimens.
- To optimize a method for enhancing signal-to-noise ratio in historical biological samples.
- To enable the study of long-term fish physiology and environmental responses.
Main Methods:
- Tested sodium borohydride (SBH), citrate buffer, and combinations on fish tissues fixed with various agents (paraformaldehyde, formaldehyde, ethanol, glutaraldehyde).
- Applied the most effective technique, SBH, to archival fish specimens (up to 65 years old).
- Performed IHC targeting Na+/K+-ATPase (NKA) on gill and skin tissues, followed by confocal fluorescence microscopy.
Main Results:
- Sodium borohydride (SBH) proved most effective in quenching autofluorescence.
- Successfully immunostained NKA in ionocytes of diverse archival fish specimens.
- Enabled clear imaging and characterization of NKA-rich ionocytes in historical samples.
Conclusions:
- The SBH-based method significantly improves IHC on archival specimens by reducing autofluorescence.
- This technique unlocks the potential of natural history collections for historical biological research.
- Facilitates understanding of fish adaptation to environmental changes over time.
Abstract:
Immunohistochemistry (IHC) is a powerful biochemical technique that uses antibodies to specifically label and visualize proteins of interests within biological samples. However, fluid-preserved specimens within natural history collection often use fixatives and protocols that induce high background signal (autofluorescence), which hampers IHC as it produces low signal-to-noise ratio. Here, we explored techniques to reduce autofluorescence using sodium borohydride (SBH), citrate buffer, and their combination on fish tissue preserved with paraformaldehyde, formaldehyde, ethanol, and glutaraldehyde. We found SBH was the most effective quenching technique, and applied this pretreatment to the gill or skin of 10 different archival fishes - including specimens that had been preserved in formaldehyde or ethanol for up to 65 and 37 years, respectively. The enzyme Na+/K+-ATPase (NKA) was successfully immunostained and imaged using confocal fluorescence microscopy, allowing for the identification and characterization of NKA-rich ionocytes essential for fish ionic and acid-base homeostasis. Altogether, our SBH-based method facilitates the use of IHC on archival samples, and unlocks the historical record on fish biological responses to environmental factors (such as climate change) using specimens from natural history collections that were preserved decades to centuries ago.
