anti β actin rabbit mab  (LI-COR)

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: hfNCSC-sEVs are taken up by PCs in vitro and enhance their proliferation and migration. (A) Primary cultures of hfNCSCs were established from male Sprague–Dawley rats. (B) Immunofluorescence staining of the neural crest cell marker p75 (red) and the stem cell marker nestin (green) in hfNCSCs, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. (C) Western blot analysis demonstrated the presence of surface markers (cluster of differentiation [CD]9, CD81, and tumor susceptibility gene 101 protein [TSG101]) and the absence of an endoplasmic reticulum marker (calnexin) in hfNCSC-sEVs. (D) Nanoparticle tracking analysis was used to quantify the concentration and size distribution of hfNCSC-sEVs. (E) Transmission electron microscopy was used to visualize the characteristic morphology of hfNCSC-sEVs. (F) Immunofluorescence staining indicated that the third-generation PCs cultured in vitro were positive for claudin-1, zonula occludens 1 (ZO1), and glucose transporter 1 (GLUT1) but negative for S100, with DAPI staining marking the nuclei. (G) The internalization of PKH26-labeled hfNCSC-sEVs (red) by ZO1-positive PCs (green) was visualized using immunofluorescence staining, with DAPI staining to mark the nuclei. (H) The Cell Counting Kit-8 assay was used to evaluate the cell viability of PCs across concentrations of 0, 2 × 10 8 , 5 × 10 8 , and 10 × 10 8 particles/mL hfNCSC-sEVs at 3, 5, and 7 days of in vitro culture ( n = 5 per group). (I) The Transwell assay was used to quantify the number of migrating PCs at 6, 12, and 18 hours post-treatment with the aforementioned concentrations of hfNCSC-sEVs, in in vitro culture ( n = 6 per group). (J) Western blot and (K) statistical analyses revealed the relative protein expression levels of proliferating cell nuclear antigen (PCNA) and vimentin in PCs from the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for H and I; Student’s t -test for K). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; GLUT1: glucose transporter 1; hfNCSCs: hair follicle neural crest stem cells; ns: not significant; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: In Vitro, Migration, Immunofluorescence, Staining, Marker, Western Blot, Concentration Assay, Transmission Assay, Electron Microscopy, Cell Culture, Labeling, Cell Counting, Transwell Assay, Expressing, Saline, Comparison, CCK-8 Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs augments cell proliferation and migration by enhancing HAS2 expression in PCs. (A, B) Western blot (A) and statistical analyses (B) revealed the relative protein expression levels of HAS2, proliferating cell nuclear antigen (PCNA), and vimentin in PCs across the –/–, –/si- Has2 , hfNCSC-sEVs/–, and hfNCSC-sEVs/si- Has2 groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (C, D) The wound healing assay (C) and statistical analysis (D) demonstrated the migration rates of PCs in the aforementioned groups ( n = 3 per group). (E) The Cell Counting Kit-8 assay was used to assess cell viability in PCs across the same groups on day 5 of in vitro culture ( n = 5 per group). (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of HAS2, PCNA, and vimentin in PCs treated with phosphate-buffered saline (PBS), hfNCSC-sEVs, or hfNCSC-sEVs + miR-21-5p inhibitor on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (H–J) Immunofluorescence staining visualized the expression of HAS2 (red) and 5-ethynyl-2′-deoxyuridine (EdU; green) in PCs (H), and statistical analysis revealed the integrated optical density (IOD) of zonula occludens 1 (ZO1; I) and the cell proliferation rates (J) in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 of in vitro culture ( n = 3 per group). (K, L) Western blot (K) and statistical analyses (L) showed the relative protein expression levels of HAS2, PCNA, and vimentin in regenerated tissue from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 post-operation (normalized to β-tubulin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, E, G, I, J, and L). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; EdU: 5-ethynyl-2′-deoxyuridine; HAS2: hyaluronan synthase 2; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

Techniques: Migration, Expressing, Western Blot, In Vitro, Wound Healing Assay, Cell Counting, Saline, Immunofluorescence, Staining, Comparison, CCK-8 Assay

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A and B , High (A, KRT5+, >40% stained neoplastic cells, n = 37) and low (B, KRT5-, n = 160) frequency of KRT5 expressing cancer cells. C , Kaplan-Meier survival analysis of HGSC patients stratified according to frequency of KRT5 expressing cells (P=0.0015). A and B, Elite ABC method. Hematoxylin counterstaining. Scale bar, 100 µm.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A , Structure of Lenti-KRT5mCherry (L-KRT5mCherry). B , Co-localization of KRT5 immunostaining (KRT5, green) and mCherry (L-KRT5) expression (magenta). Orange, overlay. Counterstaining with DAPI (blue). Scale bar, 60 µm. C , Quantification of cells expressing both KRT5 and L-KRT5 (KRT5+ L-KRT5+), or only KRT5 (KRT5+) or L-KRT5+. D , Experimental design of isolation of cells expressing both L-KRT5mCherry and L-hUbC-GFP or L-hUbC-GFP alone. E, PCR detection of L-hUbC-GFP (GFP) and L-KRT5mCherry (mCherry) DNA in both KRT5+ (K5+) and KRT5- (K5-) cells. All error bars denote s.d.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Immunostaining, Expressing, Isolation

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A and B , Organoids derived from SKOV3 cells expressing both lentiviruses (A, GFP and mCherry, orange) or GFP alone (B, green). Scale bar, 100 µm. C , Quantification of KRT5+ (blue symbols, pink bars) and KRT5- (pink symbols, yellow bars) cancer organoids in 6 consecutive passages. All error bars denote s.d. D, Volume of tumors formed by serially diluted (1 x 10 5 , 1 x 10 4 , 1 x 10 3 ) of KRT5+ and KRT5- cells after their s.c. transplantation into different flanks of NSG mice. KRT5-group did not form tumors. E and F , mCherry (E) and KRT5 (F) expression in KRT5+ cell derived xenografts. Elite ABC method. Hematoxylin counterstaining. Scale bar, 60 µm. All error bars denote s.d. G. Live microscopy of cells were isolated by FACS based on their expression of GFP (green) and mCherry (magenta) after coinfection with Lenti-UbC-GFP and Lenti-KRT5mCherry. Orange, Overlay. Individual frames of live microscopy. Scale bar, 60 µm.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Derivative Assay, Expressing, Transplantation Assay, Microscopy, Isolation

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A. Effect of cisplatin and doxorubicin on KRT5+ and KRT5- cells. Manual count (cell viability, upper row) and MTT assay (absorbance at 590 nm, bottom row) of KRT5+ and KRT5- cells after 72 hours of treating with cisplatin and doxorubicin at different concentrations. B and C . Representative confocal images of primary HGSC organoids (B) and quantification of organoid frequency (B, left image) and size (C, right image) after treatment with different concentrations of cisplatin for 72 hours. C, Immunofluorescence for KRT5 (green). Counterstaining with DAPI (blue). Scale bar, 50 µm for all images. All error bars denote s.d.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: MTT Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A . Heat map depicting the top 200 differentially expressed genes in KRT5+ (A2, A1 and A4) and KRT5- (A7, A6, and A5) cells. Note the expression of SPP1 (osteopontin) in KRT5- cells (arrow). B and C . DAVID analysis for functional annotation of terms associated with KRT5+ (B) and KRT5- (C) cells.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Expressing, Functional Assay

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A and B. Quantification of cells expressing KRT5 and/or SPP1 cells within human HGSC cases from single-cell RNA sequencing. Significance by Mann-Whitney U test. C. RT-PCR analysis of SPP1 expression in KRT5+ and KRT5- subpopulations of SKOV3 cells. D and E, KRT5 (green) and OPN (red) expression in HGSC (D) and primary HGSC organoid (E). Double immunofluorescence, counterstaining with DAPI (blue). Scale bar, (D) 60 µm, E (40 µm). F and G , OPN treatment increases frequency (F) and size (G) of HGSC organoids (n=3). H - K . Effect of cisplatin on frequency (H and J) and size (I and K) of organoids either transduced with SPP1 shRNA (H and I) or treated with OPN (I and K). All organoids were measured 72 hours after treating with cisplatin at different concentrations. All error bars denote s.d.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Expressing, RNA Sequencing, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Transduction, shRNA

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic of laser-cut PDMS microwell array reversibly assembled on a polystyrene petri dish and workflow of enzymatically amplified silver metallization using Biotin-BSA and poly-HRP-SA. (b) Cell-phone image of silver metallized wells after assay. (c) Biotin-BSA dilution curve obtained from cell-phone image silver darkness quantification in ImageJ. (d) Assay stack for initial biotinyl tyramide signal amplification testing using biotinylated anti-IFN-γ as capture. (e) Dilution curve of silver darkness of increasing biotinylated anti-IFN-γ as target and 10 µg/mL, 1 µg/mL, and 0 µg/mL biotinyl tyramide. All assays were repeated three (n=3) times and fit lines are four-parameter logistic sigmoidal curves.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Amplification

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic of immunoassay for cytokine detection. (b) Initial LOD optimization for the IFN-γ assay. (c-e) IFN-γ (n=2), IL-2 (n=5), TNF-α (n=2) dilution curves using recombinant cytokines (f-h) Correlation plots of ELISA absorbance values at 450 nm versus silver darkness values for IFN-γ (f), TNF-α (g), and IL-2 (h). All fit lines are four-parameter logistic sigmoidal curves.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: (a) Schematic demonstrating the stimulation groups and controls. Dilution curves for IFN-γ (b-c), IL-2 (d-e), and TNF-α (f-g) in pooled LTB+ and LTB-clinical supernatant samples. Clinical samples consisted of NIL (negative control), Ag1, Ag2, and Mitogen (positive control) groups (n=2).

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: Negative Control, Positive Control

Journal: bioRxiv

Article Title: Inexpensive High-Throughput Multiplexed Cytokine Detection for Tuberculosis Diagnostics Using Amplified Enzymatic Metallization

doi: 10.64898/2026.01.27.700981

Figure Lengend Snippet: Individual participant silver darkness values for IFN-γ (a), IL-2 (b), and TNF-α (c) detection from NIL, Ag1, Ag2, and Mitogen samples of IGRA+ and IGRA-patients using a sample dilution of 1:10. Average of n=2 repeats was plotted for each sample.

Article Snippet: Recombinant human IFN-γ protein (11725-HNAS), biotinylated anti-human IFN-γ antibody (11725-R209-B), anti-human IFN-γ antibody (11725-R238), HRP-conjugated anti-human IFN-γ antibody (11725-R209-H), recombinant human TNF-α protein (10602-HNAE), biotinylated anti-human TNF-α antibody (10602-MM08-B), and anti-human TNF-α antibody (10602-MM01) were obtained from SinoBiological.

Techniques: