Anti-CD3 抗体 (ab5690)

CD3 and active Caspase 3 populations 72 hrs after mouse aortic allograft

C57Bl/6 donor aortic allografts were transplanted into Balb/C recipient mice (N = 3 per treatment) and followed up at 72hrs. Compared to saline, Serp-2 but not CrmA treatment reduced caspase 3 activity (panels A-C; p<0.0224). Neither protein treatment significantly reduced CD3+ T cells (panels D-F).

T cells in central nervous system during late disseminated infection

A-E) Representative epifluorescence images of T cells (CD3), blood vessels (CD31), and nucleated cells (DAPI) in the brain, dura mater, and pia mater. (A) Epifluorescence images described from left to right. CD3 shown in FITC channel; CD31+ blood vessels shown in TRITC channel; nucleated cells shown in DAPI channel; merged image showing CD3+ cell associated with pia mater within the commissure of the isocortex. (B) T cells within the lymphatic-like vascular region of the sagittal sinus in the dura mater. (C) T cell associated with a blood vessel in the vasculature of the brain choroid plexus. (D) T cell associated with blood vessel in the dura mater. (E) T cell in extravascular region of the dura mater.

(After Figure 3 of Dirvan et al)

IHC image of CD3 staining in a formalin fixed, paraffin embedded normal human tonsil tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) for 20 mins. The section was then incubated with ab5690 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab5690 staining CD3 (green) in mouse lymph node tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with acetone/alcohol and blocked with 1% BSA for 1 hour at 23°C. Samples were incubated with primary antibody (1/250 in 1% BSA) for 12 hours at 4°C. An Alexa Fluor^® 568-conjugated goat anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.

CD8 (red) was labelled with ab22378 followed by an Alexa Fluor^® 647-conjugated goat anti-rabbit IgG.

CD3+ CD8+ are yellow.

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Lanes 1 - 6: Merged signal (red and green). Green – ab5690 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab5690 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling CD3 with ab5690 at 2µl/ml. Slides were steamed in IHC epitope retrieval solution for 35 minutes and then cooled for 20 minutes. Samples were incubated with the primary antibody at room temperature for 1 hour, incubated with a biotinylated secondary antibody for 30 minutes followed by HRP-Streptavidin for 30 minutes. Developed with DAB chromogen substrate for 5-10 minutes. Counter stained with hematoxylin. Magnification: 10X.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling CD3 with ab5690 at 2µl/ml. Slides were steamed in IHC epitope retrieval solution for 35 minutes and then cooled for 20 minutes. Samples were incubated with the primary antibody at room temperature for 1 hour, incubated with a biotinylated secondary antibody for 30 minutes followed by HRP-Streptavidin for 30 minutes. Developed with DAB chromogen substrate for 5-10 minutes. Counter stained with hematoxylin. Magnification: 40X.

No positive immunostaining for CD3ε, a pan T cell marker (ab5690), was detected in the corneas of scrambled siRNA-treated mice (A) at 5 days p.i. In contrast, positive immunostaining (red) was observed in the peripheral cornea of HIF-1α silenced animals (B). The control sections shown in (C) and (D) were immunostained with species-specific IgG and were positive for SYTOX Green nuclear stain only. Images shown are representative of three independent experiments each with three mice per group. Magnification = 180×; inset = 335×.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue labelling CD3 with ab5690 at 2µl/ml. Slides were steamed in IHC epitope retrieval solution for 35 minutes and then cooled for 20 minutes. Samples were incubated with the primary antibody at room temperature for 1 hour, incubated with a biotinylated secondary antibody for 30 minutes followed by HRP-Streptavidin for 30 minutes. Developed with DAB chromogen substrate for 5-10 minutes. Counter stained with hematoxylin. Magnification: 10X.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue labelling CD3 with ab5690 at 2µl/ml. Slides were steamed in IHC epitope retrieval solution for 35 minutes and then cooled for 20 minutes. Samples were incubated with the primary antibody at room temperature for 1 hour, incubated with a biotinylated secondary antibody for 30 minutes followed by HRP-Streptavidin for 30 minutes. Developed with DAB chromogen substrate for 5-10 minutes. Counter stained with hematoxylin. Magnification: 40X.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue showing no expression of CD3 when labelled with ab5690 at 2µl/ml. Slides were steamed in IHC epitope retrieval solution for 35 minutes and then cooled for 20 minutes. Samples were incubated with the primary antibody at room temperature for 1 hour, incubated with a biotinylated secondary antibody for 30 minutes followed by HRP-Streptavidin for 30 minutes. Developed with DAB chromogen substrate for 5-10 minutes. Counter stained with hematoxylin. Magnification: 40X.

ab5690 staining CD3 in the lymphatic nodule of rat spleen by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer pH 6.0. Samples were then blocked with 1% BSA for 25 minutes at 20°C followed by incubation with the primary antibody at a 1/100 dilution for 1 hour at 20°C. A biotin-conjugated goat anti-rat polyclonal was used undiluted as the secondary antibody. CD3 positive cells are mainly distributed in PALS (T-cell region), while sporadic CD3 positive cells are identified in lymphoid follicle and germinal center (B-cell region).

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