Figure 1: CD68 target protein structure.

CD68 Target Introduction

Protein Function

• CD68 is a transmembrane glycoprotein that is highly expressed in monocytes and macrophages.
• Although it is lowly expressed in hematopoietic cell lines, CD68 can be induced by PMA/TPA.
• It can bind to tissue and organ-specific lectins or selectins, allowing macrophage subpopulations to home to specific sites.
• It is expressed in many tumor cell lines and can attach to selectins on the endothelium, promoting their spread to secondary sites. Therefore, CD68 is a potential marker for tumor metastasis.

Protein Localization

• CD68 is localized to the cell membrane, endosomal membrane, and lysosomal membrane.

Figure 2: CD68 ICC experimental result image, Anti-CD68 antibody [EPR20545] (ab213363). Green: CD68, Red: Tubulin, Blue: DAPI.

Isoforms & post-translational modifications

• Human (P34810): Isoforms 1-3: 32-37 kDa (predicted)
• Mouse (P31996): Isoforms 1-2: 34-35 kDa (predicted)
• Rat (Q4FZY1): 35 kDa (predicted)
• Presence of N- and O-glycosylation modifications

WB experiment tips

Precautions

• Due to the highly glycosylated modification of CD68 protein, the actual detected band size is between 110-150 kDa, which is different from the theoretical value of 32-37 kDa.

Positive control

• Human tonsils
• Fetal liver
• THP-1 and U937 whole cell lysates

Example of results

Figure 3: WB-Anti-CD68 antibody [EPR20545] (ab213363).

Lane 1: 20 µg human fetal liver lysate
Lane 2: 20 µg human tonsil lysate
Lane 3: 20 µg human fetal spleen lysate
Lane 4: 20 µg THP-1 (human monocytic leukemia cell line) whole cell lysate
Lane 5: 20 µg U937 (human tissue cell lymphoma cell line) whole cell lysate

Primary antibody:

Lanes 1-3 & 5: Anti-CD68 antibody [EPR20545] (ab213363), diluted 1/1000.
Lane 4: Anti-CD68 antibody [EPR20545] (ab213363), diluted 1/5000.
Secondary antibody: Goat anti-rabbit HRP-conjugated antibody (ab97051), diluted 1/100000.
Predicted band size: 37 kDa
Detected band size: 110 kDa

IHC experiment tips

Precautions

• No need for antigen retrieval on acetone-fixed frozen sections.
• CD68 is mainly expressed on the lysosomal membrane of myeloid cells, and staining is observed in the cytoplasm.

Positive controls

• Human tonsil tissue
• Cervical cancer tissue

Example of results

Figure 4: IHC - Anti-CD68 Antibody [EPR20545] (ab213363)

Sample name: Formalin/PFA-fixed human tonsil tissue paraffin sections
Primary antibody: Anti-CD68 Antibody [EPR20545] (ab213363), diluted at a concentration of 1/8000.
Secondary antibody: HRP-labeled goat anti-rabbit secondary antibody.
Antigen retrieval method: Tris/EDTA pH 9.0 antigen retrieval solution was used.
Description: Cytoplasmic staining of macrophages in human tonsil tissue can be observed.

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, fixing at room temperature for 18-24 hours is more appropriate.
2. Excessive fixation will close antigenic epitopes. Although antigen retrieval will expose some of the epitopes, if the tissue fixation time is very long (such as more than a week), there will still be no signal after antigen retrieval.

Antigen retrieval:

1. The conditions for antigen retrieval need to be optimized. We recommend performing retrieval at 110℃ for 15 minutes in a pressure cooker.

Antibody incubation:

1. When conducting the initial experiment, please optimize the working concentration of the antibody according to the product instructions.
2. If the experiment allows, please also perform a control experiment with only secondary antibody and no primary antibody.

References

1. E Kunisch, R Fuhrmann, A Roth, R Winter, W Lungershausen, R W Kinne. Macrophage specificity of three anti-CD68 monoclonal antibodies (KP1, EBM11, and PGM1) widely used for immunohistochemistry and flow cytometry. Ann Rheum Dis. 2004 Jul;63(7):774-84. doi: 10.1136/ard.2003.013029.
2. Mariella Caffo, Gerardo Caruso, Antonino Germanò, Salvatore Galatioto, Francesco Meli, Francesco Tomasello. CD68 and CR3/43 immunohistochemical expression in secretory meningiomas. Neurosurgery. 2005 Sep;57(3):551-7; discussion 551-7. doi: 10.1227/01.neu.0000170440.32720.77.
3. Dimitry A Chistiakov, Murry C Killingsworth, Veronika A Myasoedova, Alexander N Orekhov, Yuri V Bobryshev. CD68/macrosialin: not just a histochemical marker. Lab Invest. 2017 Jan;97(1):4-13. doi: 10.1038/labinvest.2016.116. Epub 2016 Nov 21.