Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Abstract

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.

Key words::

• capsule
• fibrous tissue
• gold chloride
• labrum
• nerve endings
• shoulder
• stain

Acknowledgments

We thank Daniel Sisk for assisting with staining specimens, Dr. Takako Chickenji, Mayo Clinic, Rochester, MN, for providing critical knowledge of cryoprotectants for sectioning the labrum, and Damon Mar for building the device for flattening and freezing tissue. This investigation was supported by grants T32HD057850 and HD02528 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Partial support was received from the Marc A. and Elinor J. Asher Orthopedic Research Endowment.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.