Figure1: CD31 target protein structure.

CD31 Target Introduction

Protein Function

• CD31 is a cell adhesion molecule that promotes leukocyte transendothelial migration.
• CD31-CD31 homophilic interaction determines most of the biological functions of CD31.
• The heterophilic interaction between CD31 and CD177 promotes neutrophil transendothelial migration.
• CD31 can participate in cell adhesion through homophilic transendothelial interaction.
• Stimulation with fMLP or CXCL8/IL-8 downregulates the expression of CD31 on the surface of neutrophils.

Protein Expression

• Expressed on platelets and leukocytes, mainly concentrated at the endothelial cell border.
• Expressed in human umbilical vein endothelial cells (HUVECs) (protein level).
• Expressed in neutrophils (protein level).
• Isoform 1 is predominantly expressed in all tested tissues.
• Isoform 2 is only detected in the trachea.
• Isoform 5 is only detected in the lungs.
• Isoform 4 is detected in all examined tissues, with the strongest expression in the heart.
• Isoform 6 is expressed in the brain, testes, ovaries, platelet surface, human umbilical vein endothelial cells (HUVECs), Jurkat T-cell leukemia, human erythroleukemia (HEL), and U-937 tissue cell lymphoma cell line (protein level).

Protein Localization

• CD31 is localized to the cell membrane, cell junctions, and lateral border recycling compartment (LBRC), and can circulate from the LBRC to the junctions of resting endothelial cells.

Figure 2: CD31 ICC experimental result image, Anti-CD31 antibody [EPR3094] (ab76533). Green: CD31, Red: Tubulin, Blue: DAPI.

Isoforms & post-translation modifications

• Human (P16284): Isoforms 1-6: 79-83 kDa (predicted)
• Mouse (Q08481): Isoforms 1-4: 70-81 kDa (predicted)
• Rat (Q3SWT0): 76 kDa (predicted)
• CD31 protein has multiple glycosylation sites.
• After activation of cells by Src kinase, phosphorylation occurs on Ser and Tyr residues.
• Palmitoylation mediated by ZDHHC21 is necessary for the expression of CD31 on the surface of endothelial cells and its enrichment in membrane rafts.

WB experiment tips

Precautions

• Due to the post-translational modification of CD31 protein, the actual detected human isoform protein band size is around 130 kDa, which is different from the theoretical value of 79-83 kDa.
• CD31 has expression specificity, and there are obvious differences in expression levels in different tissues/cells. We recommend using positive controls.

Positive controls

• HUVEC (human umbilical vein endothelial cells) whole cell lysate
• Jurkat whole cell lysate (ab7899)

Negative control

• NIH/3T3 whole cell lysate (ab7179)

Example of results

Figure 3: WB-Anti-CD31 antibody [RM1006] (ab281583).

Lane 1: 20 μg HUVEC cell lysate
Lane 2: 20 μg bEnd.3 cell lysate
Lane 3: 20 μg NIH/3T3 cell lysate
Lane 4: 20 μg rat lung tissue lysate

Primary antibody: Anti-CD31 antibody [RM1006] (ab281583), diluted at 1/1000 concentration.
Secondary antibody: HRP-conjugated goat anti-rabbit IgG antibody (ab97051), diluted at 1:100000 concentration.
Predicted band size: 82 kDa
Detected band size: 125 kDa

Key control points

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

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.

Transferring:

1. We strongly recommend using Coomassie Brilliant Blue staining after transferring to determine the success of the transfer.

IHC experiment tips

Precautions

• CD31 is mainly expressed in vascular endothelial cells, we recommend testing tissues that are rich in blood vessels.
• Staining enrichment phenomenon occurs between the boundaries of endothelial cells.

Positive control

• Human umbilical vein endothelial cells (HUVECs)
• Skin, kidney, tonsil, prostate, liver and heart.

Example of Results

Figure 4: IHC - Anti-CD31 Antibody [EPR17259] (ab182981)

Sample name: Formalin/PFA-fixed human lung tissue paraffin sections
Primary antibody: Anti-CD31 Antibody [EPR17259] (ab182981), diluted at 1:2000 concentration.
Secondary antibody: HRP-labeled goat anti-rabbit secondary antibody.
Antigen retrieval method: Heat-induced antigen retrieval using Tris/EDTA pH 9.0 antigen retrieval solution.
Other: Staining of human lung endothelial cell membrane (PMID: 16234507), counterstained with hematoxylin.

Key control points

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

Sample fixation:

1. The time for sample fixation depends on the size of the tissue block and the type of tissue, but for most samples, such as fixation with 4% PFA, fixing at room temperature for 18-24 hours is more appropriate.
2. Insufficient fixation can result in higher signal at the edges of the sample and weaker or even no signal in the center.
3. Excessive fixation can block antigenic epitopes. Although antigen retrieval can expose some of the epitopes, if the tissue fixation time is very long (such as more than a week), there may still be no signal after antigen retrieval.

Antigen retrieval:

1. When performing immunohistochemistry on paraffin sections, we recommend using a pressure cooker for heat-induced antigen retrieval. You can try fixing the sections at 110°C for 15 minutes. After retrieval, let it cool naturally and avoid putting it in cold water to prevent section detachment due to sudden temperature drop.
2. When performing immunohistochemistry on frozen sections, if the samples have been fixed with aldehydes for 18-24 hours, you can try using a microwave for short-term retrieval or using enzymes for antigen retrieval. However, please optimize the enzyme concentration and retrieval time to avoid damaging the tissue morphology of the sections.

Blocking:

1. If subsequent detection is performed using an HRP conjugate, please use 3% hydrogen peroxide to treat the sections for 10 minutes to block endogenous peroxidase.
2. If using fluorescently labeled secondary antibodies for the experiment, we recommend using a blocking solution containing 1% BSA and a final concentration of 0.3 M glycine to quench the autofluorescence caused by aldehyde groups.
3. Before incubating with the primary antibody, it is necessary to block with serum and avoid selecting a blocking solution from the same species as the host. The source of the serum can be selected based on the host of the secondary antibody. For example, if the secondary antibody is Goat Anti-Rabbit IgG H&L (HRP polymer) or Goat Anti-Mouse IgG H&L (HRP polymer), goat serum can be used as the blocking solution.
4. Pay attention to the species of the host for the primary antibody and the species of the sample being detected. For example, when detecting mouse tissue with a primary antibody derived from mouse, non-specific staining is prone to occur. In this case, appropriate IgG blocking reagents can be added to prevent binding with endogenous IgG, thereby reducing non-specific staining.

References

1. Panida Lertkiatmongkol , Danying Liao, Heng Mei, Yu Hu, Peter J Newman. Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31). Curr Opin Hematol. 2016 May;23(3):253-9. doi: 10.1097/MOH.0000000000000239.
2. Jason L.HornickMD, PhD. 1 - Introduction: Tumor Classification and Immunohistochemistry. Practical Soft Tissue Pathology: a Diagnostic Approach (Second Edition). 2019, Pages 1-7. doi: 10.1016/B978-0-323-49714-5.00001-6