Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies

Abstract

Cell transplant and gene therapies are promising approaches to many disorders of the nervous system. In studies involving cell transplants to the brain or nervous system, expression of green fluorescent protein (GFP) is commonly used to label cells, allowing their identification and histological assessment even after long post-operative survival times. Techniques employing viral tracing or reporter genes also commonly use GFP to label cells. Here, we document the presence of a subpopulation of green autofluorescent cells in the cortex and hippocampus of formaldehyde fixed, cryosectioned rat brains aged 3–9 months. Using standard microscopic fluorescence imaging techniques, we acquired clear images of green autofluorescent cells, complete with extensive processes, which appear to be well integrated into the host tissue. Treatment of brain sections with sodium borohydride followed by cupric sulfate in ammonium acetate buffer reduced background and cellular autofluorescence throughout sections but, especially in hippocampus, did not eliminate considerable green fluorescence in a subset of neurons. This autofluorescence was weak and would therefore pose a problem only when cells weakly express GFP or when few labeled cells survive. We suggest that investigators be aware of the potential for false positives, especially if the cells expressing GFP are expected to migrate widely from the transplant site. Parallel sections from naïve brains should regularly be processed and imaged alongside experimental brain sections, and anti-GFP immunohistochemistry should be performed to ensure that true GFP+ signals are imaged instead of endogenous autofluorescent neurons.

Spontaneous development of Alzheimer's disease‐associated brain pathology in a Shugoshin‐1 mouse cohesinopathy model

“…After deparaffinization, antigen retrieval, sodium borohydride treatment, CuSO 4 treatment, and blocking, the slides were treated with primary antibodies for 16 hr, then with secondary fluorescent antibodies for 1 hr, followed by brief DAPI staining and sealing with antifade. Sodium borohydride and CuSO 4 were used to minimize autofluorescence by Shiff‐base and by Lipofuscin, respectively (Schnell, Staines, & Wessendorf, 1999; Spitzer, Sammons, & Price, 2011). …”

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

“…Unspecific cellular/tissue autofluorescence with broad excitation/emission may arise from lipofuscin accumulation with aging, typically causing a speckled cytosolic staining (41,49,50). Other potential sources of autofluorescence, which even persist tissue fixation, are NADH and serotonin (50).…”

“…Other potential sources of autofluorescence, which even persist tissue fixation, are NADH and serotonin (50). As such background fluorescence may interfere with roGFP1 imaging, we compared nontransgenic wild-type CCD, chargecoupled device; CTX, cortex; DG, dentate gyrus; HP, hippocampus; IC, inferior colliculus; roGFPc, cytosolically expressed roGFP; STR, striatum; S1, primary somatosensory cortex; TH, thalamus; VIS, visual cortex; Str.…”

Triple-Labeling Whole-Mount In Situ Hybridization Method for Analysis of Overlapping Gene Expression in Brain Tissue with High Level of Autofluorescence

“…Autofluorescence, either intrinsic or induced by fixation reagents and tissue processing, may mask specific fluorescent signals or be mistaken for fluorescent labels [16,17]. Autofluorescence of neural tissue often complicates the use of the green fluorescent protein (GFP) [18]. More importantly for our research, animals with spinal cord transection showed significant autofluorescence of cortical, brainstem, and spinal cord neurons, which interferes with identification of labeling [19,20].…”