Formaldehyde-fixed, non-permeabilized mouse colon tissue stained for CD4 with ab183685 (30 mins at a 1/500 dilution) in immunohistochemical analysis. A Rabbit polyclonal HRP conjugate was used as the secondary.

Heat mediated antigen retrieval buffer/enzyme used: pH 9.0 EDTA.

Blocking step: 1% ab64226 for 10 mins at RT.

All lanes : Anti-CD4 antibody [EPR19514] (ab183685) at 1/2000 dilution

Lane 1 : Mouse thymus lysate
Lane 2 : Mouse liver tissue lysate
Lane 3 : Mouse brain tissue lysate
Lane 4 : Mouse kidney tissue lysate
Lane 5 : Raw264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate
Lane 6 : NIH/3T3 (mouse embryo) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

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

The expression profile observed is consistent with what has been described in the literature (PMID: 2155425).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1:3 seconds; Lanes 2,3,4,5 and 6:30 seconds

 

IHC image of CD4 staining in a section of frozen normal Mouse Spleen.

The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab183685 (Rabbit monoclonal [EPR19514] to CD4) at 1/200 and ab243840 at 1µg/ml, to show the distinct staining of B cells and T cells. The section was then incubated with ab150165 (Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preabsorbed, (Shown in green) 1/1000) and ab150080 (Goat Anti-Rabbit IgG H&L (Alexa Fluor®594) (Shown in red) 1/1000) for 1 hour at room temperature. The secondary-only control insert image is taken from an identical assay without primary antibody. DAPI was used to stain the cell nuclei (blue). The section was then mounted using Fluoromount®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen tissue labeling CD4 with ab183685 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The result showed membrane staining on mouse spleen.

The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

Antigen retrieval: Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

Paraformaldehyde-fixed, 0.1% Tween 0.3% Triton in PBS permeabilized mouse liver tissue stained for CD4 with ab183685 (12 hours, 4°C at a 1/100 dilution) in immunohistochemical analysis. A Donkey anti Rabbit IgG (H+L) AlexaFluor^®647 was used as the secondary at a 1/500 dilution (red).

Blocking step: 1% BSA for 12 hours at 4°C.

Formalin-fixed, 0.2% Triton-X permeabilized mouse Eo771 mammary tumor tissue stained for CD4 with ab183685 (18 hours, 4°C at a 1/1000 dilution) in immunohistochemical analysis. A Goat anti Rabbit IgG (H&L) polyclonal AlexaFluor^®488 conjugate was used as the secondary (green).

Heat mediated antigen retrieval buffer/enzyme used: Citrate buffer.

Blocking step: 4% BSA for 1 hour at 25°C.

Formaldehyde-fixed, non-permeabilized mouse brain tissue stained for CD4 with ab183685 (14 hours, 4°C at a 1/1000 dilution) in immunohistochemical analysis. A Goat anti Rabbit IgG monoclonal HRP conjugate was used as the secondary at a 1/200 dilution.

Blocking step: 5% normal goat serum for 1 hour at 26°C.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Formaldehyde-fixed, paraffin-embedded mouse lung tissue stained for CD4 with ab183685 (18 hours, 4°C at a 1/1000 dilution) in immunohistochemical analysis. A Goat anti Rabbit IgG polyclonal HRP conjugate was used as the secondary at a 1/250 dilution (red).

Heat mediated antigen retrieval buffer/enzyme used: Citrate buffer pH 6.0.

Blocking step: 10% serum for 1 hour at 25°C.

Formaldehyde-fixed, 3% hydrogen peroxide permeabilized B16F10 (melanoma) syngeneic mouse tumor tissue stained for CD4 with ab183685 (36mins, 37°C at a 1/1000 dilution) in immunohistochemical analysis. A Goat anti Rabbit IgG polyclonal HRP conjugate was used as the secondary.

Heat mediated antigen retrieval buffer/enzyme used: Tris/EDTA.

Formaldehyde-fixed, non-permeabilized mouse spleen tissue stained for CD4 with ab183685 (30mins at a 1/1000 dilution) in immunohistochemical analysis. A Goat polyclonal HRP conjugate was used as the secondary.

Heat mediated antigen retrieval buffer/enzyme used: Leica ER2 solution.

Formaldehyde-fixed mouse colon, chronic mucosal inflammation tissue stained for CD4 with ab183685 (90 mins at a 1/1000 dilution) in immunohistochemical analysis. A Goat polyclonal biotin conjugate was used as the secondary.

Heat mediated antigen retrieval buffer/enzyme used: High pH CC1.

Blocking step: 1% BSA for 40 mins at 24°C.

CD4 was immunoprecipitated from 1mg of Mouse thymus whole cell lysate with ab183685 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab183685 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Mouse thymus whole cell lysate, 10µg (Input).

Lane 2: ab183685 IP in Mouse thymus whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730)  instead of ab183685 in Mouse thymus whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds.

10% NBF-fixed, paraffin-embedded mouse spleen tissue stained for CD4 using ab183685 at 1/2000 dilution in immunohistochemical analysis, followed by Goat anti-Rabbit IgG Alexa Fluor^® 647.

See Abreview

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling CD4 with ab183685 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative on mouse cerebrum.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Formaldehyde-fixed, paraffin-embedded mouse spleen tissue stained for CD4 using ab183685 at 1/2000 dilution in immunohistochemical analysis, followed by Goat anti Rabbit IgG Biotin.

See Abreview

Formaldehyde-fixed, paraffin-embedded mouse thymus tissue stained for CD4 using ab183685 at 1/500 dilution in immunohistochemical analysis.

See Abreview