Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

Abstract

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions:

Pre-Exposure to Environmental Enrichment Protects against Learning and Memory Deficits Caused by Infrasound Exposure

“…We used FJC (Chemicon, Temecula, CA, USA), which can specifically stain degenerating neurons in the CNS subject to various neurotoxin insults and neurological diseases [ 35 , 36 ].…”

“…Brain sections containing the hippocampus were prepared as in Section 2.9 . FJC staining was carried out using the following standard procedures [ 35 , 36 ]: (1) pretreatment with an alcohol-sodium hydroxide mixture. The sections were immersed in a solution containing 1% sodium hydroxide in 80% alcohol for 5 min, followed by 70% alcohol and distilled water each for 2 min.…”

Mitochondrial Aldehyde Dehydrogenase 2 (ALDH2) Protects against Binge Alcohol-Mediated Gut and Brain Injury

“…To investigate the brain damage, we prepared cryosections of the mid-brain for staining with Fluoro-Jade C (F.-J.C.), which is widely used to identify degenerative neuronal cells, including apoptotic and necrotic cells [39]. However, F.-J.C. may also detect non-neuronal cells, as reported [40]. In our confocal microscopy analysis, the DAPI-stained slides were used as positive controls to stain all neuronal cells.…”

A peptide-neurotensin conjugate that crosses the blood-brain barrier induces pharmacological hypothermia associated with anticonvulsant, neuroprotective and anti-inflammatory properties following status epilepticus in mice

“…To estimate cell degeneration, we performed FJC histo-staining (Chemicon, Temecula, CA, USA) on sections of dorsal hippocampus from SHAM, SE, SE + VH-N412, SE + NT(8-13) and SE + DZP animals according to the standard protocol of Schmued et al (2005) and Bian et al (2007). This histostaining does not depend on the mode of cell death (Ikenari et al, 2021). The specimens were analyzed using a confocal microscope and images were acquired using Zen software (Zeiss, LSM 700, Jena Germany), and processed using Adobe Photoshop and ImageJ softwares.…”