title: Improved gold chloride procedure for nerve staining in whole mounts of rat corneas.
abstract: The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryoprotective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at -70 C in O.C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO4-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO4-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO4-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O.C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced non-specific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohistochemical procedures where the reaction products would be obscured by background staining.
authors.affiliation: Section on Pathophysiology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Jacot
authors.forename: J P
authors.initials: JP
authors.lastname: Glover
authors.forename: W G
authors.initials: WG
authors.lastname: Robison
mesh_terms: D000818:Animals; D003315:Cornea / Q000294:innervation*; D003451:Cryoprotective Agents; D015353:Eye Enucleation; D017612:Gold Compounds; D006651:Histocytochemistry; D016613:Plastic Embedding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*; D000516:alpha-Amylases / Q000378:metabolism
chemical_list: D003451:Cryoprotective Agents; D017612:Gold Compounds; C038016:gold chloride; D000516:alpha-Amylases