Immunofluorescence quality of human brain tissue fixed with solutions used in gross anatomy laboratories

Abstract Brain banks provide small tissue samples fixed in neutral-buffered-formalin (NBF), but human anatomy teaching laboratories could provide full brains fixed with solutions that are more appropriate for gross anatomy such as a saturated salt solution (SSS) or an alcohol-formaldehyde solution (AFS). Advanced aging and prolonged exposure to aldehydes are known to enhance brain tissue autofluorescence (AF), limiting the efficacy of immunofluorescence (IF) procedures. We have previously shown by IF staining the antigenicity preservation in mouse brains fixed with the three solutions. We now aimed to compare the quality of IF staining in human brains fixed with SSS, AFS and NBF. In addition, we compared the efficiency of AF quenching methods, namely the application of SudanBlackB (SBB) and the treatment of sections with sodium borohydride (NaBH 4 ). Blocks of neocortex were extracted from 18 brains (NBF=6, SSS=6, AFS=6) and cut into 40µm sections. Neurons (anti-NeuN, AlexaFluor-488) and astrocytes (anti-GFAP, AlexaFluor-555) were revealed with IF after an antigen retrieval protocol, while two treatments (SBB or NaBH 4 ) were used to quench AF. We then assessed the degree of AF (criteria: background or cell AF) and the immunostaining quality with excitation wavelengths of 488nm, 555nm and 647nm. Brains fixed with all three solutions showed well-labeled astrocytes, whereas neurons weren’t always stained, but this was not associated to the fixative solution. The overall AF intensity was similar in sections from brains fixed with all three solutions. Finally, the SBB treatment was the most effective at reducing AF in all specimens. Given the similarity in AF and antigenicity assessment across the three solutions, we conclude that brains fixed with SSS and AFS could be good alternatives for NBF-fixed specimens in the context of IF experiments processed with a SBB protocol. Highlights - Immunofluorescence staining is feasible in brains fixed with anatomy labs solutions - GFAP is less affected by fixation than NeuN - Autofluorescence can be reduced by Sudan Black treatment