anti rabbit igg starbright blue 700 fluorescent secondary antibody  (Bio-Rad)

Journal: bioRxiv

Article Title: Neuroligin-2 is ubiquitinated by Nedd4l to control developmental astrocyte morphogenesis

doi: 10.64898/2025.12.15.694023

Figure Lengend Snippet: A) Schematic of AAV constructs and the experimental strategy of in vivo BioID to identify NL interactome in vivo . Each biological replicate contained two pooled cortices, 3 biological replicates per condition. B) Cartoon depiction of Neuroligin iBioID constructs in astrocytes or neurons. C-D). Representative cortical images of Astrocytic Turbo BirA (C) or Astrocytic NL2 Turbo BirA (D) injected animals. Stained with Streptavidin-594 (Strept), HA and Sox9 to determine astrocyte specificity. Scale bars: column image = 100µm; inset = 20µm. E) Representative western blot of iBioID immunoprecipitation from astrocytic Turbo vs NL2-Turbo animals demonstrates successful enrichment of biotinylated proteins in immunoprecipitation. Input is 2% of total cortex homogenate. IP is 15% of the eluate sample. Prominent bands are expected at approximately 150 and 75kDa which represent mitochondrial carboxylases [blot is overexposed to show this in the input lanes]. Resulting ‘smear’ in IP lanes indicated various-sized proteins that have been biotinylated near the bait protein. F) Venn diagram of protein interactors across astrocytic NL1, NL2, and NL3 and astrocytic versus neuronal NL2.

Article Snippet: Alexa Fluor or Streptavidin conjugated fluorescent secondaries (Invitrogen: Rabbit 405+ RRID:AB_2890548; Mouse IgG1 488 RRID:AB_2535764, Chicken 568 RRID:AB_2534098, Guinea Pig 647 RRID:AB_2535867, Rat 488 RRID:AB_2534074, Rabbit 594 RRID:AB_2534095, Streptavidin Alexa Fluor conjugate (Thermo Fisher, S32357)) were diluted in blocking buffer at 1:200 and incubated on tissue for 3 hours at room temperature.

Techniques: Construct, In Vivo, Injection, Staining, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Neuroligin-2 is ubiquitinated by Nedd4l to control developmental astrocyte morphogenesis

doi: 10.64898/2025.12.15.694023

Figure Lengend Snippet: A) Representative images of surface-stained astrocyte/neuron co-cultures. Cells were fixed with cold 4% PFA for 5 minutes, and triton was omitted from blocking steps to avoid intracellular staining. GFP and GFAP were used as negative controls. HA (magenta) signal shows membrane localization as expected. B) Representative images demonstrating effective biotinylation (Streptavidin Alexa 594, red) in HA-NL2-Turbo-PDZ but not in HA-NL2 (negative control) or HA-NL2-Turbo. Experiments were performed at least twice with at least 20 cells per condition. C) Sholl analysis quantification of images in B. Linear-mixed model reveals the main effect of condition, F(1,3)=11.01, p=1.4E-6. P-values represent Dunnett’s post-hoc with respect to shNL2 + NL2-RM. NS = not significant in Dunnett’s. N = 14-56 cells across 3 biological replicates (independent culture experiments). D). Representative cortical images of Neuronal Turbo BirA injected animals. Stained with Streptavidin-594 (Strept), HA and NeuN to determine neuronal specificity. Scale bars: column image = 100µm; inset = 20µm.

Article Snippet: Alexa Fluor or Streptavidin conjugated fluorescent secondaries (Invitrogen: Rabbit 405+ RRID:AB_2890548; Mouse IgG1 488 RRID:AB_2535764, Chicken 568 RRID:AB_2534098, Guinea Pig 647 RRID:AB_2535867, Rat 488 RRID:AB_2534074, Rabbit 594 RRID:AB_2534095, Streptavidin Alexa Fluor conjugate (Thermo Fisher, S32357)) were diluted in blocking buffer at 1:200 and incubated on tissue for 3 hours at room temperature.

Techniques: Staining, Blocking Assay, Membrane, Negative Control, Injection