cy5 secondary antibody  (Jackson Immuno)

Journal: Advanced Science

Article Title: A Biomimetic cRGDyk‐Engineered Platelet Nanoplatform for ACAD11 Delivery to Trigger Pyroptosis in Hepatocellular Carcinoma

doi: 10.1002/advs.78219

Figure Lengend Snippet: The construction and characterization of OE‐ACAD11@R‐PLT. (A) Schematic representation of the construction of OE‐ACAD11@R‐PLT. (B) The size distribution of PLT, OE‐ACAD11, cRGDyk, and OE‐ACAD11@R‐PLT. (C) The zeta potential of PLT, cRGDyk, OE‐ACAD11, and OE‐ACAD11@R‐PLT. (D) SEM images of raw PLT and OE‐ACAD11@R‐PLT. (E) The marker protein (CD62P, CD61, CD41, and GAPDH) in raw PLT and OE‐ACAD11@R‐PLT. (F) CLSM results of OE‐ACAD11@R‐PLT. Green, FITC‐labeled PLTs. Red, Cy5‐labeled cRGDyk. (G) Drug loading efficiency and encapsulation rate of OE‐ACAD11@R‐PLT.

Article Snippet: First, the cRGDyk peptide was labeled with CY5 (MCE, USA) via amino‐carboxyl coupling.

Techniques: Zeta Potential Analyzer, Marker, Labeling, Encapsulation

Journal: Bioactive Materials

Article Title: Countering postoperative immune suppression with a self-assembling dendritic cell nanovaccine

doi: 10.1016/j.bioactmat.2026.05.005

Figure Lengend Snippet: BAITs enhance antigen uptake and digestion by DCs under TGFβ stimulation. (A) Schematic illustration of real-time DC migration assay. BAITs in the lower chamber create a chemokine gradient to attract DCs from the upper chamber. (B) Kinetics of DC migration measured as cell index (n = 3; n.s. is P > 0.05, ∗∗∗∗ is P < 0.0001 by two-way ANOVA with Bonferroni post-hoc test). (C) Crystal violet staining of non-migrated DCs (upper chamber) and Calcein-AM staining of migrated DCs (lower chamber) showing enhanced DC recruitment by BAITs. (D) The antigen uptake efficiency of DCs under varying antigen concentrations with or without TGFβ (n = 5; ∗∗∗ is P < 0.001, ∗∗∗∗ is P < 0.0001 by two-way ANOVA with Bonferroni post-hoc test). (E) Heatmap of immune-related genes in DCs showing upregulation of antigen presentation and co-stimulation markers and downregulation of suppressive factors as antigen concentration increases. (F) Antigen digestion capacity of DCs assessed in the presence or absence of TGFβ (n = 8; ∗∗∗∗ is P < 0.0001 by two-tailed Student's t -test). (G) Fluorescent images showing enhanced antigen uptake in DCs mediated by BAITs (quantified on right; n = 10; ∗∗∗∗ is P < 0.0001 by two-way ANOVA with Bonferroni post-hoc test). (H) Flow cytometry analysis of antigen-positive (Cy3 + ) DCs after incubation with free antigen or BAIT (quantified on right; n = 4; ∗∗∗∗ is P < 0.0001 by two-way ANOVA with Bonferroni post-hoc test). (I) BAITs improve antigen digestion and uptake of DCs under TGFβ stimulation. DCs were first incubated with Cy5-antigens for 12 h, followed by medium replacement and subsequent treatment with Cy3-antigens or Cy3-BAITs for an additional 6 h. Data are presented as mean ± SD.

Article Snippet: For MHC I analysis, the cells were incubated with rat CoraLite Plus 488 anti-mouse LAMP1 antibody (1:200) and rabbit anti-mouse MHC I antibody (1:100) overnight at 4 °C, followed by Cy5-conjugated anti-rabbit secondary antibody (1:200) for 1 h at 37 °C.

Techniques: Migration, Staining, Immunopeptidomics, Concentration Assay, Two Tailed Test, Flow Cytometry, Incubation

Journal: CNS Neuroscience & Therapeutics

Article Title: Targeting Microglial HDAC5 Suppresses NLRP3 Inflammation via NF ‐ κB Deacetylation to Alleviate Ischemic Brain Injury

doi: 10.1002/cns.71161

Figure Lengend Snippet: Preparation and characterization of RGD‐EV:MiR‐9‐5p. (A) Western blot was used to detect the expression of the positive markers Alix and TSG101 in EVs. (B) Representative images of TTC‐stained brains from I/R model mice at 24 h after tail vein injection of PBS or EVs. (C) Quantification of infarct volume after TTC staining, n = 3. (D) Neurological deficit scores in the sham group, and I/R model mice treated with EVs or PBS. (E) RT‐PCR analysis of the levels of 10 different miRNAs in HEK293T‐derived EVs and ReN‐derived EVs ( n = 3). (F) After EV ReN was added to HT‐22 cells for 24 h, OGD experiments were performed and 5 miRNAs were detected ( n = 3). *denotes comparison with the Control group, *** p < 0.001; # denotes comparison between the OGD group and the OGD + EV ReN group, ### p < 0.001. (G) The binding sites of miR‐9‐5p on Hdac5 were predicted by TargetScan. (H) Dual‐luciferase reporter gene assay was used to verify the binding between miR‐9‐5p and Hdac5 . (I) BV2 cells were transfected with miR‐9‐5p mimics, and after 48 h, the expression of HDAC5 in the cells was analyzed by Western blot. (J) Quantification of HDAC5 ( n = 4). (K) Schematic of RGD‐EV, labeled with Cy5.5, and loaded with cholesterol‐modified miR‐9‐5p. (L, M) TEM imaging (L) and NTA (M) of RGD‐EV: MiR‐9‐5p; scale bar 200 nm. (N, O) MCAO/R mice were injected with Cy5.5‐labeled RGD‐EVs or RGD‐EV: MiR‐9‐5p, and Cy5.5 levels in the brain were analyzed 24 h later. Representative brain NIR fluorescence images (N) and quantification of intracerebral fluorescence intensity (O) are shown, n = 3. (P) After ischemia modeling in mice, tdTomato‐labeled EVs were injected into the tail vein and perfused 24 h later. Fluorescent staining was performed to analyze whether EVs had entered into microglia in the ischemic region; red indicates EVs, green indicates microglia, and the scale bar is 25 μm. (Q, R) BV2 cells were pretreated with PBS, EV (or EV: MiR‐NC, or EV: MiR‐9‐5p) for 24 h before LPS treatment. The expression of HDAC5 (Q) in the cells were determined by Western blot analysis, and quantification of HDAC5 (R), n = 3. Data are expressed as mean ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001 by one‐way ANOVA.

Article Snippet: For imaging, Cy5.5 azide (0.3 nmol; Lumiprobe, USA) was added simultaneously.

Techniques: Western Blot, Expressing, Staining, Injection, Reverse Transcription Polymerase Chain Reaction, Analysis, Derivative Assay, Comparison, Control, Binding Assay, Luciferase, Reporter Gene Assay, Transfection, Labeling, Modification, Imaging, Fluorescence