widefield microscope  (Nikon)

Journal: bioRxiv

Article Title: Neuronal overexpression of Nrf2 reduces dystrophic neurites in 5XFAD Alzheimer’s disease model mice

doi: 10.64898/2026.03.16.711596

Figure Lengend Snippet: ( A ) Representative brain sections from 5XFAD and non-transgenic (non-TG) mice transduced with Nrf2+GFP or GFP-only AAVs and stained with DAPI (blue), BACE1 (Green) and Thiazine Red (ThR, red) to mark plaques and imaged by widefield immunofluorescence microscopy. Size bar = 500μm ( B ) Percent area covered by BACE immunostaining in 5XFAD cortex is elevated, which has a non-significant downward trend (p=0.07) caused by neuronal Nrf2 overexpression ( C ) Percent area covered by BACE immunostaining in 5XFAD hippocampus is elevated, which is significantly reduced by neuronal Nrf2 overexpression. 2-way ANOVA with Sidak’s multiple comparisons test * p<0.05, ** p<0.01, **** p<0.0001 ( D ) Representative single plane confocal images of amyloid plaques (red, Thiazine red, *= plaque core) surrounded by dystrophic neurites immunostained for BACE1, LAMP1, and p-tau181 (left to right, green) in GFP-only or Nrf2+GFP AAV transduced 5XFAD mice. Note that in the right panel of images from Nrf2 overexpressing mice, dystrophic neurites are smaller, fewer and of less overall plaque coverage than upper panel (arrows). Size bar = 10μm. ( E-G ) Quantification of the ratio of dystrophic neurite proteins (BACE, E; LAMP1, F; tau p181, G) to amyloid plaque core (Thiazine red, E, G; Aβ42, F) in cortex of GFP-only or Nrf2+GFP AAV transduced 5XFAD mice. In all cases, the ratios are reduced in neuronal Nrf2 overexpressing 5XFAD mice, indicating reduced dystrophic neurites for a given amount of amyloid. ( H-J ) Quantification of the mean areas of BACE1, LAMP1 or tau p181 immuno-positive structures around individual amyloid plaques in cortex. For all three proteins, the average areas of immunostaining are reduced in Nrf2 overexpressing mice. ( E-J ) quantification from 10x Ti2 widefield images, using NIS Elements, as described in methods. * p<0.05, ** p<0.01, two tailed t-test. NB: Images are false colored for viewing. Imaging channels were as follows: DAPI, NeuN – 405nm, ThR, Aβ42 – 568nm, BACE1, Aβ42, p181 - 647nm. Non-TG GFP n= 5 (3F,3M), non-TG Nrf2+GFP n= 6 (4F, 2M), 5XFAD GFP n= 10 (5F, 5M), 5XFAD GFP+Nrf2 n= 10 (5F, 5M)

Article Snippet: Images were acquired on a Nikon Ti2 Eclipse widefield microscope with a 10x objective, using the NIS Elements software high content method to capture and tile whole sections.

Techniques: Transgenic Assay, Transduction, Staining, Immunofluorescence, Microscopy, Immunostaining, Over Expression, Two Tailed Test, Imaging