Human peripheral blood lymphocytes stained with unpurified ab133616 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (unpurified ab133616, 1/100 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD4 with purified ab133616 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

All lanes : Anti-CD4 antibody [EPR6855] (ab133616) at 1/1000 dilution (unpurified)

Lane 1 : THP-1 cell lysate
Lane 2 : Human fetal thymus lysate
Lane 3 : Human tonsil lysate
Lane 4 : Human lymph node lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : HRP labelled goat anti-rabbit at 1/2000 dilution
Lanes 2-4 : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa

Immunocytochemistry analysis of THP-1 (Human monocytic leukemia monocyte) labeling CD4 with purified ab133616 at 1/100 dilution. Cells were fixed with 100% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.30 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative control: PBS instead of the primary antibody.

HBZ is preferentially expressed in CD4+ T cells of HAM/TSP patient PH1624

Confocal microscopy analysis of PBMC from HAM/TSP patient PH1624. (A) co-staining with the 4D4-F3 anti-HBZ mAb followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and with the anti-CD4 mAb followed by Alexa Fluor 488-conjugated goat-anti-rabbit IgG antibody (green); upper panels, extended field; lower panels, enlarged field focused on the single cell depicted in the square of the left upper panel and positive for both CD4 and HBZ.

CD4 was detected using ab133616 at 1/100 dilution.

From Figure 6A of Baratella et al.

Baratelle et al PLoS Negl Trop Dis. 2017 Jan; 11(1): e0005285. Published online 2017 Jan 17. doi: 10.1371/journal.pntd.0005285

Reproduced under  Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thymoma tissue labelling CD4 with purified ab133616 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling CD4 with ab133616 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Anti-CD4 antibody [EPR6855] (ab133616)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD4 with ab133616 at a dilution of 1:500. Heat mediated antigen retrieval was performed using AR9 antigen retrieval solution, and microwave treatment for 15 min at 20% power. Anti-Rabbit/Mouse HRP polymer (PerkinElmer Opal Polymer HRP Ms Plus Rb) was used as secondary antibody. Opal tyramide amplification was performed using Opal 520 fluorophore. Counterstained with DAPI stain. Image scanned with Vectra 3.0 and analyzed via Phenochart software.
This image was courteously provided by Dr. Houssein Abdul Sater, Georgia Cancer Center.

Paraffin-embedded human spleen tissue stained for CD4 using ab133616 at 1/500 dilution in immunohistochemical analysis.

See Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD4 with unpurified ab133616 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling CD4 with unpurified ab133616.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling CD4 with unpurified ab133616.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thymoma tissue labelling CD4 with unpurified ab133616.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Negative control: no staining on human cerebrum.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum showing no staining CD4 with purified ab133616 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Counterstained with hematoxylin.

Negative control: no staining on human pancreas.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreas showing no staining CD4 with purified ab133616 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Counterstained with hematoxylin.

All lanes : Anti-CD4 antibody [EPR6855] (ab133616) at 1/5000 dilution (purified)

Lane 1 : Human fetal thymus tissue lysate
Lane 2 : Human tonsil tissue lysate
Lane 3 : THP-1 cell lysate
Lane 4 : HuT-78 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa

Blocking and dilution buffer: 5% NFDM/TBST.