nuclei counterstaining with hoechst  (Thermo Fisher)

Journal: Molecular Neurobiology

Article Title: Intracrine VEGF Signaling Is Required for Adult Hippocampal Neural Stem Cell Maintenance and Vascular Proximity

doi: 10.1007/s12035-025-04861-1

Figure Lengend Snippet: VEGFR2 and VEGF are expressed in the same NSCs. a Kdr and Vegfa in situ hybridization co-labeled with GFAP antibody in adult mouse DG. Scale bar represents 10 µm. b Percent of putative RGL-NSCs that are Kdr + , Vegfa + , or Kdr + / Vegfa + by in situ hybridization coupled with immunolabeling for GFAP. N = 4 mice; mean ± SEM. c Representative immunofluorescent image of VEGFR2 immunolabeling with Nestin in a VEGF-GFP mouse. Arrowheads show VEGFR2 + /VEGFGFP + /Nestin + co-expression. Scale bar represents 20 µm. d Representative 3D renderings of z-stack images of VEGFA and VEGFR2 immunoreactivity in the ER (PDI) or Golgi (RCAS1, Syntaxin) in permeabilized cultured NSCs. Hoechst labels nuclei. Major lines in image boxes are 10 µm apart

Article Snippet: The following day, after three rinses in PBS, cells were incubated in secondary antibodies diluted in blocking solution for 2 h. If a biotinylated secondary was used, a fluorophore-conjugated tertiary was applied for 1 h diluted in PBS before rinsing and nuclei counterstaining with Hoechst (10 min, 1:2000 in PBS) (Invitrogen).

Techniques: In Situ Hybridization, Labeling, Immunolabeling, Expressing, Cell Culture

Journal: Molecular Neurobiology

Article Title: Intracrine VEGF Signaling Is Required for Adult Hippocampal Neural Stem Cell Maintenance and Vascular Proximity

doi: 10.1007/s12035-025-04861-1

Figure Lengend Snippet: Cell autonomous VEGF signaling maintains NSCs in culture. a Diagram of the neighbor rescue experiment. b , c VEGF concentration (pg/ml) in NSC conditioned media after treatment with high MOI ( b ) or low MOI ( c ) mCherry or mCherryCre lentivirus. N = 3 wells/grp; mean ± SEM plus individual wells shown. d Representative immunofluorescent images of BrdU + cultured Vegfa lox/lox and WT NSCs (Hoechst + ) after lentiviral infection (mCherry + ). Chevrons indicate BrdU + mCherry + Hoechst + NSCs. e Percent of mCherry + or mCherry − Vegfa lox/lox and WT NSCs that were BrdU + after low MOI mCherryCre and mCherryonly lentiviral infection. N = 3/grp/exp, 3 exps. Mean ± SEM shown. f Representative images of cultured Vegfa lox/lox NSCs (brightfield) after lentiviral infection (mCherry + ) after 1 and 4 passages. Images taken immediately after passage, when counting was performed. g Representative images of cultured WT NSCs (brightfield) after lentiviral infection (mCherry + ) after 1 and 4 passages. Images taken immediately after passage, when counting was performed. h Percent of mCherry + Vegfa lox/lox and WT NSCs after infection with low MOI mCherryCre or mCherryonly lentiviral vectors. N = 3/grp/exp, 2 exps; mean ± SEM. Scale bars represent d 10 µm and f , g 50 µm. * p < 0.05; ** p < 0.01; **** p < 0.0001

Article Snippet: The following day, after three rinses in PBS, cells were incubated in secondary antibodies diluted in blocking solution for 2 h. If a biotinylated secondary was used, a fluorophore-conjugated tertiary was applied for 1 h diluted in PBS before rinsing and nuclei counterstaining with Hoechst (10 min, 1:2000 in PBS) (Invitrogen).

Techniques: Concentration Assay, Cell Culture, Infection

Journal: Frontiers in Cellular Neuroscience

Article Title: EAAT2 Expression in the Hippocampus, Subiculum, Entorhinal Cortex and Superior Temporal Gyrus in Alzheimer’s Disease

doi: 10.3389/fncel.2021.702824

Figure Lengend Snippet: EAAT2 expression in the superior temporal gyrus (STG). Photomicrographs of layer III of the STG showing EAAT2 ( A,D , red), GFAP ( B,E , green), and EAAT2 overlaid with GFAP and NeuN (magenta; C,F ) immunoreactivity for a representative control ( A–C , H123) and Alzheimer’s disease (AD) ( D–F , Az90) case. In controls, EAAT2 labeling is more localized to membranes of astrocytic stem processes, while in AD the staining is weaker on astrocytic main branches ( A–F , arrowheads), but more labeling is likely localized to fine astrocytic branches in the neuropil. Cell nuclei are labeled with Hoechst nuclei counterstain ( C,F , blue). Scale bar (A–F) = 10 μm.

Article Snippet: Sections were then washed for 10 min in PBST before incubation for 35 min at RT with Hoechst nuclei counterstain (1:10,000, 33342, RRID:AB_10626776 , Invitrogen) diluted in PBS.

Techniques: Expressing, Labeling, Staining