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.

Neuroanatomical distribution of mechanoreceptors in the human cadaveric shoulder capsule and labrum

“…We have outlined a brief overview of the staining protocol; however, a more detailed version has been published (Witherspoon et al. ).…”

“…For this reason, we developed a method that permitted flattening of the tissue along the contact plane for proper sectioning (Witherspoon et al. ). Sections were then mounted to gelatin subbed slides and the slides were dehydrated using 70% ethanol, 100% ethanol and two xylene baths, sequentially.…”

“…One of the cadaveric shoulders was found to be missing a labrum. Capsule and labrum tissues were dissected from the glenohumeral joint as described elsewhere (Witherspoon et al 2014). Briefly, the specimens were dissected from the most common region of injury, the anteroinferior region, at 3-6 o'clock, and from the least common region of injury, the posteroinferior region, at 6-9 o'clock as shown in Fig.…”

“…The gold chloride staining technique was chosen because it reliably stains mechanoreceptors in glenoid capsules and labrums. We have outlined a brief overview of the staining protocol; however, a more detailed version has been published (Witherspoon et al 2014).…”

“…The embedding process used for capsular specimens was more complex than that for labral specimens. To section the capsules in the plane of the articular contact surface, we developed a novel embedding process in which the capsules were first flattened using a custom clamping device and then frozen (Witherspoon et al 2014).…”