Anti-CD3 epsilon/CD3E Antibody Picoband®

Western blot analysis of CD3E using anti-CD3E antibody (PB9093).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Jurkat whole cell lysates,
Lane 2: human Jurkat whole cell lysates,
Lane 3: human Hut-78 whole cell lysates,
Lane 4: human Hut-78 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-CD3E antigen affinity purified polyclonal antibody (Catalog # PB9093) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CD3E at approximately 23 kDa. The expected band size for CD3E is at 23 kDa.

IHC analysis of CD3 Epsilon using anti-CD3 Epsilon antibody (PB9093).
CD3 Epsilon was detected in paraffin-embedded section of rat spleen tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD3 Epsilon Antibody (PB9093) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CD3 Epsilon using anti-CD3 Epsilon antibody (PB9093).
CD3 Epsilon was detected in paraffin-embedded section of human tonsil tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD3 Epsilon Antibody (PB9093) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CD3 Epsilon using anti-CD3 Epsilon antibody (PB9093).
CD3 Epsilon was detected in paraffin-embedded section of mouse spleen tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD3 Epsilon Antibody (PB9093) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IF analysis of CD3E and CD20 using anti-CD3E antibody (PB9093) and anti-CD20 antibody (M03780-5).
CD3E and CD20 was detected in a paraffin-embedded section of human tonsil tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/mL rabbit anti-CD3E antibody (PB9093) and mouse anti-CD20 antibody (M03780-5) overnight at 4°C. DyLight®550 Conjugated Goat Anti-Rabbit IgG (BA1135), DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

IF analysis of CD3E using anti-CD3E antibody (PB9093).
CD3E was detected in a paraffin-embedded section of mouse 4T1 cell xenograft tumor tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:200 rabbit anti-CD3E Antibody (PB9093) overnight at 4°C. DyLight 550-conjugated Goat Anti-Rabbit was used as secondary antibody incubated for 1 hour at RT. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

IHC analysis of CD3 Epsilon using anti-CD3 Epsilon antibody (PB9093).
CD3 Epsilon was detected in frozen section of rat spleen tissues. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD3 Epsilon Antibody (PB9093) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IHC analysis of CD3 Epsilon using anti-CD3 Epsilon antibody (PB9093).
CD3 Epsilon was detected in frozen section of mouse spleen tissues. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD3 Epsilon Antibody (PB9093) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

IF analysis of CD3E and CD68 using anti-CD3E antibody (PB9093) and anti-CD68 antibody (M00602)
CD3E and CD68 was detected in paraffin-embedded section of human tonsil tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution ) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/mL rabbit anti-CD3E Antibody (PB9093) and mouse anti CD68 Antibody(M00602) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) and Biotin conjugated goat anti-rabbit IgG (BA1003) were used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The tissue section was developed using Cy3 Conjugated Avidin (BA1037). The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

a Schematic diagram of BCP-GNCs-mediated macrophage polarization and DC maturation by releasing IL-4 and DXMS. b IF staining of Arg1 (red) and iNOS (green) of macrophage, CD83 (red) and CD40 (green) of DC and nuclei (blue) after macrophages or DCs seeded on the BCP with 690 nm far-red or 808 nm NIR stimulating the release of PBS (BCP-PBS), 690 nm far-red stimulating the release of IL-4 (BCP-IL4) or 808 nm NIR stimulating the release of DXMS (BCP-DXMS) for 24 h. Scale bar = 5 μm. c, d Semiquantification of positively stained cells in ( b ). n = 5, ** P < 0.01, *** P < 0.001. e Flowchart of time course for irradiating BCP-GNC in vivo. f–h IHC staining of M1, M2 macrophages (iNOS and Arg1), mature DCs (CD40), T cells (CD3), MSCs and osteoblasts (CD146, Runx2) under the BCP-GNCs implant in vivo. Scale bar = 100 μm. Red arrow, positive cells. M, material. i – k Semiquantification of positively stained cells in (E, F, G). n = 5, * P < 0.05, ** P < 0.01, **** P < 0.0001. l H&E and Masson staining of implant area of BCP-Con and BCP-GNCs in vivo after 4 weeks (4 W) (the red dash line shows the new bone formation area; NB, new bone; M, material). m Semiquantification of new bone area in ( k ). n = 5, **** P < 0.0001
Index in PubMed under a CC BY license. PMID: 34138183

a Implementation strategy of clodronate on macrophages depletion. b H&E and Masson staining of BCP implant area treating with PBS or clodronate in vivo after implant 4 weeks (the red dash line shows the new bone formation area; NB, new bone; M, material). Scale bar = 100 μm. c IHC staining of M2 macrophages (Arg1) and osteoblasts (Runx2, Col1a1) under the BCP implant treating with PBS or clodronate in vivo. Scale bar = 100 μm. Red arrow, positive cells. M, material. d – g Semiquantification of new bone area and positively stained cells in ( b, c ). n = 5, *** P < 0.001, **** P < 0.0001. h Implementation strategy of diphtheria toxin (DT) on DCs depletion. i H&E and Masson staining of BCP implant area treating with PBS or DT in vivo after implant 4 weeks (the red dash line shows the new bone formation area; NB, new bone; M, material). Scale bar = 100 μm. j IHC staining of T cells (CD3) and osteoblasts (Runx2, Col1a1) under the BCP implant treating with PBS or DT in vivo. Scale bar = 100 μm. Red arrow, positive cells. M, material. k – n Semiquantification of new bone area and positively stained cells in ( i, j ). n = 5, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
Index in PubMed under a CC BY license. PMID: 34138183

OSBP translocates from Golgi to PM via interacting with ORP4L. a Electron micrographs of ORP4L KI and wild-type T-cells, HepG2 and 293 T cells. Scale bar, 100 nm. Black triangles present in dividual data points for n = 20 cells from one or cell line. The central mark indicates the median, the bottom and top edges of the box indicate the interquartile range, and the whiskers represent the maximum and minimum point. One-way ANOVA test with a confidence interval of 95% was used to compute statistics. b Co-immunoprecipitation analysis of ORP4L binding to OSBP in ORP4L KI T-cells with or without anti-CD3 stimulation. c The localization of OSBP in ORP4L KI T-cells. Scale bar, 10 μm. d The localization of OSBP in ORP4L KI and wild-type T-cells with or without anti-CD3 stimulation. Scale bar, 10 μm. e Western blot analysis of OSBP protein levels in PM and Golgi of ORP4L KI or wild-type T-cells. f A schematic representation describing the BiFC technique. g Interactions between ORP4L/pVn-C1 and OSBP/pVc-C1 in ORP4L KI T-cells as determined by BiFC. Scar bar, 10 μm. h The localization of OSBP in ORP4L KI T-cells upon ORP4L knockdown. Scar bar, 10 μm. i Western blot analysis of OSBP protein levels in PM and Golgi of ORP4L KI T-cells upon ORP4L knockdown. j The localization of overexpressed wild-type OSBP or OSBP ( △ ORP4L) in ORP4L KI T-cells. Scar bar, 10 μm. k Western blot analysis of OSBP protein levels in PM and Golgi of ORP4L KI T-cells with or without VAPA knockdown. l Western blot analysis of overexpressed wild-type OSBP or OSBP( △ FFAT) protein levels in the PM and Golgi of ORP4L KI T-cells. Confocal microscopy and blot images are representative of n = 3 biological replicates with similar results. The same experiments were repeated twice in T-cells from two mice. In ( c , d , h and j ), the ration of OSBP fluorescence density from n = 10 cells from one between PM and Golgi are shown below. Source data are provided as a Source Data file.
Index in PubMed under a CC BY license. PMID: 35906240

Pathological findings of T-cell leukemia in ORP4L KI mice. a Kaplan–Meier comparative survival analysis of ORP4L KI and WT littermate mice ( n = 20 mice per group, Log-rank test). b White blood cell count on peripheral blood of ORP4L KI and WT littermate mice. Black triangles present in dividual data points for n = 15 (ORP4L KI) and n = 12 (WT) mice. c The percentage of indicated cells in peripheral blood of ORP4L KI and WT littermate mice. Black triangles present in dividual data points for n = 10 mice. d Percentage of CD25 + Foxp3 + or CD25 + CCR4 + cells in peripheral blood of ORP4L KI and WT littermate mice. Black triangles present in dividual data points for n = 8 (ORP4L KI) and n = 6 (WT) mice. e Flow cytometry analysis of Foxp3 and CCR4 expression in ORP4L KI and WT littermate mice. The same experiments were repeated twice in two mice. f Representative blood smears of ORP4L KI and WT littermate mice ( n = 3 mice). Scale bars, 10 μm. g , h Splenomegaly and hepatomegaly in ORP4L KI mice. The right panels illustrate the organ weights. Black triangles present in dividual data points for n = 10 (ORP4L KI) and n = 6 (WT) mice. Scale bars, 1 cm. i Representative H&E-staining of organs in ORP4L KI mice. Immunohistochemical anti-CD3 antibody staining (inserts) of the tissues is shown. Scale bars, 100 μm. j Gross and histological findings of splenomegaly in B-NDG mice after transplantation of spleen cells from ORP4L KI mice. Black triangles present in dividual data points for n = 6 mice. Scale bars, 1 cm. k Representative H&E and anti-CD3 antibody staining of spleen of j . Scale bars, 100 μm. Images of i and k are representative of n = 3 biological replicates with similar results. The same experiments were repeated twice in two mice. In each box plot, the central mark indicates the median, the bottom and top edges of the box indicate the interquartile range, and the whiskers represent the maximum and minimum point. Two-tailed unpaired t -test with a confidence interval of 95% was used to compute statistics. P -values are indicated in the figures. Source data are provided as a Source Data file.
Index in PubMed under a CC BY license. PMID: 35906240