class iii β tubulin tuj 1  (R&D Systems)

Journal: Nature cell biology

Article Title: Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer's disease.

doi: 10.1038/s41556-025-01623-y

Figure Lengend Snippet: Fig. 1 | Transdifferentiation of human adult fibroblasts into neurons reveals signatures of ageing and AD. a, Human dermal fibroblasts were collected from healthy young and aged, aged/sAD and fAD-PSEN1 donors. Fibroblasts and tNeurons were used for a variety of experiments and the findings were validated in post-mortem brain tissue as well as CSF. b, Levels of DNA damage (left) measured as the number of the nuclear foci of γ-H2AX immunofluorescence (right) in human fibroblasts. White dotted lines outline cell nuclei. Images are representative of three independent experiments; n = 248 young, 304 aged and 252 aged/sAD cells from three donors. Scale bar, 50 μm. c, Age- and AD-related epigenetic alterations. Immunofluorescence of the histone modifications H3K9me3 (left) and H4K16ac (right) in human fibroblasts was measured. Data are from four donors and three independent experiments; H3K9me3, n = 252 young, 241 aged and 237 aged/sAD cells; H4K16ac, n = 157 young, 167 aged and 141 aged/sAD cells. d, Neuronal transdifferentiation efficiency. Representative images of human fibroblasts immunostained for S100A4 and Vimentin, and tNeurons immunostained for Tuj1, GAP43, MAP2 and NeuN with 4,6-diamidino-

Article Snippet: The tNeurons were permeabilized with 0.3% Triton X-100 in PBS for 5 min at room temperature or cold methanol on ice for 20 min and blocked with pre-warmed 5% BSA in PBS at room temperature for 45 min. Next, the tNeurons were incubated overnight in a moisture chamber at 4 °C with the following primary antibodies in 5% BSA: anti-ASC/PYCARD Nature Cell Biology (1:100; Santa Cruz, sc-514414), anti-GAP-43 (1:300; Novus Biologicals, NB300-143), anti-MAP2 (1:1,000; BioLegend, 822501), anti-NeuN (1:500; Abcam, ab177487), anti-NLRP3 (1:200; R&D systems, MAB7578), anti-p62/SQSTM1 (1:500; Proteintech, 18420-1-AP), anti-amyloid-β (1:100; Cell Signaling Technologies, 8243), anti-amyloid-β (1–42) (1:200; Enzo Life Sciences, ADI-905-804-100), anti-APP-CTF (1:200; BioLegend, 802803), anti-HGS (1:200; GeneTex, GTX101718), anti-Hsp27 (HspB1; 1:100; Proteintech, 18284-1-AP), anti-Hsp70 (1:1,000; Abcam, ab45133), anti-LC3B (1:100; Cell Signaling Technologies, 2775), anti-TDP-43 (1:1,000; Proteintech, 12892-1-AP), anti-pTau (AT8) (1:100; Thermo Fisher Scientific, MN1020), anti-pTau (S262) (1:200; FUJIFILM WAKO, 010-27123), anti-pTDP-43 Ser409/410 (1:300; Cosmo Bio, CAC-TIP-PTD-M01 and 1:200; BioLegend, 829901), anti-synapsin-1 (1:200; Abcam, ab64581), anti-tau (1:100; Aves Labs) and anti-β tubulin III (Tuj1) (1:500; Neuromics, CH23005 and 1:1,000; BioLegend, 801201).

Techniques: Immunofluorescence

Journal: JCI insight

Article Title: Gut mucosal cells transfer α-synuclein to the vagus nerve.

doi: 10.1172/jci.insight.172192

Figure Lengend Snippet: Figure 2. Human A53T α-synuclein protein transfers from gut cells to adjoining vagal neurons. (A) Intestinal organoids were prepared from an SNCAA53T mouse in which CCK-containing cells express enhanced green fluorescent protein (eGFP), and vagal nodose ganglia neurons were isolated from an Snca–/– mouse lacking endogenous α-synuclein. (B) Representative images of organoids and neurons grown in coculture for 5 days, with eGFP-positive cells (green) in the organoid and β-tubulin III (Tuj1, cyan) highlighting neuronal processes. (C) Representative high-magnification α-synuclein (red) staining of an eGFP-positive EEC. Red arrow indicates localization to a PGP9.5-positive (cyan) process in an Snca–/– mouse neuron. (D and E) Representative images with neuron-specific β-tubulin III (cyan). Surface and (adjacent) intracellular confocal slices are shown. Scale bars are 30 μm for B, 3 μm for C, and 5 μm for D and E.

Article Snippet: Primary antibodies used for immunostaining included rabbit CCK (73), rabbit α-synuclein (Abcam catalog ab138501, RRID:AB_2537217, at 1:1,000), guinea pig PGP9.5 (Abcam catalog ab10410, RRID:AB_297150, at 1:100), chick β-tubulin III (Tuj1; Neuromics catalog CH23005, RRID:AB_2210684, at 1:100), and chick GFP (Abcam catalog ab13970, RRID:AB_300798, at 1:1,000).

Techniques: Isolation, Staining