Figure 1: Structure of the human T cell receptor-CD3 complex protein.

CD3ε Target Introduction

Protein Function

• CD3 is a T-cell marker. The TCR:CD3 complex is a multi-subunit protein complex consisting of a TCR (T-cell receptor) αβ heterodimer that binds to antigens, and four peptide chains of CD3 (including ε, γ, δ, and ζ), forming three dimers: εγ, εδ, and ζζ. The cytoplasmic domains of all CD3 peptide chains contain immunoreceptor tyrosine-based activation motifs (ITAMs).
• The TCR:CD3 complex is involved in T-cell recognition of antigens, and the activation signals generated by TCR recognition of antigens are transduced by CD3 into the T-cell.
• CD3ε plays an important role in the proper development of T-cells. The TCR-CD3 complex initiates assembly by forming two heterodimers, CD3δ/CD3ε and CD3γ/CD3ε. Through endocytic sequences present in the cytoplasmic region of CD3ε, it participates in the internalization and downregulation of the TCR-CD3 complex on the cell surface.

Protein Localization

• Cell membrane

Figure 2: ICC experimental results of the CD3 epsilon target, using the recombinant Anti-CD3 epsilon antibody [EPR22667-12] product (ab231775). Green: CD3 epsilon, Red: alpha Tubulin, Blue: DAPI.

Isoforms & Post-translational modifications

• Human (P07766): 23 kDa (predicted)
• Mouse (P22646): 21 kDa (predicted)
• Rat (D4A5M2): 21 kDa (predicted)
• Phosphorylation

IHC experiment tips

Precautions

• The TCR-CD3 complex is present on the surface of T lymphocytes and plays an important role in adaptive immune responses.

Positive control

• Rat spleen tissue
• Human tonsil tissue
• Mouse lymph node tissue

Example results

Figure 3: IHC- Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD3 antibody [SP7] product (ab16669).

Sample name: Rat spleen tissue.
Primary antibody: Used ab16669 primary antibody, diluted at a concentration of 1:100.
Secondary antibody: HRP conjugated compact polymer system.
Antigen retrieval method: Heat-induced antigen retrieval with sodium citrate buffer (pH 6) for 20 minutes.

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample fixation:

1. The fixation time of the sample depends on the size of the tissue block and the type of tissue, but for most samples, 4% PFA fixation at room temperature for 18-24 hours is more appropriate.
2. Insufficient fixation can cause the tissue sections to have strong staining at the edges and no signal in the middle.
3. Excessive fixation will block antigenic epitopes. Although antigen retrieval will expose some of the epitopes, if the tissue fixation time is very long (more than a week), there may still be no signal after antigen retrieval.

Antigen retrieval:

1. The conditions for antigen retrieval need to be optimized. When performing immunohistochemistry experiments on paraffin sections, try fixing the slides in a pressure cooker at 110°C for 15 minutes.
2. In general, antigen retrieval is not necessary for frozen sections. If PFA is used to fix the sample for 18-24 hours for frozen sections, try using an enzyme antigen retrieval method, and never use heat-induced antigen retrieval.

Blocking:

1. If fluorescence methods are used to detect signals, it is recommended to add 0.3 M glycine to the blocking solution to quench autofluorescence.
2. If HRP conjugates are used for detection, use 3% hydrogen peroxide to treat the sections for 10 minutes to block endogenous peroxidase.

Antibody incubation:

1. For the first experiment, please choose the appropriate working concentration of the antibody according to the antibody datasheet.
2. If the experiment allows, perform a control experiment with only secondary antibody and no primary antibody.

References

1. Jatuporn Ngoenkam, Wolfgang W Schamel, Sutatip Pongcharoen. Selected signalling proteins recruited to the T-cell receptor-CD3 complex. Immunology. 2018. 153(1):42-50. doi: 10.1111/imm.12809.
2. Michael S Kuhns, Mark M Davis, K Christopher Garcia. Deconstructing the form and function of the TCR/CD3 complex. Immunity. 2006. 24(2):133-9. doi: 10.1016/j.immuni.2006.01.006.
3. Clifford S Guy, Dario A A Vignali. Organization of proximal signal initiation at the TCR:CD3 complex. Immunol Rev. 2009. 232(1):7-21. doi: 10.1111/j.1600-065X.2009.00843.x.
4. A Borroto, J Lama, F Niedergang, A Dautry-Varsat, B Alarcón, A Alcover. The CD3 epsilon subunit of the TCR contains endocytosis signals. J Immunol. 1999. 163(1):25-31.
5. Nadia Martin-Blanco, Daniel Jiménez Teja, Gabriel Bretones, Aldo Borroto, Michael Caraballo, Isabella Screpanti, Javier León, Balbino Alarcón, Matilde Canelles. CD3ε recruits Numb to promote TCR degradation. Int Immunol. 2016.  28(3):127-37. doi: 10.1093/intimm/dxv060.