EM1901-95

CD68 Mouse Monoclonal Antibody [A3C4]

cat.: EM1901-95

Product Type:	Mouse monoclonal IgG1, primary antibodies
Species reactivity:	Human
Applications:	WB, IHC-P, FC, mIHC
Clonality:	Monoclonal
Clone number:	A3C4

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*PBS (pH7.4), 0.2% BSA, 50% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	2ug/ul
Purification:	Protein G affinity purified.
Molecular weight:	Predicted band size: 37 kDa.
Isotype:	IgG1
Immunogen:	Synthetic peptide within human CD68 aa 320-354.
Positive control:	A431 cell lysate, THP-1 cell lysate, U-937 cell lysate, human tonsil tissue, human lung tissue, THP-1, human pancreatic carcinoma.
Subcellular location:	Cell membrane. Endosome membrane, lysosome membrane.
Recommended Dilutions: WB IHC-P FC mIHC	1:1,000-1:2,000 1:200-1:1,000 1:1,000 1:3,000
Uniprot #:	SwissProt: P34810 Human
Alternative names:	CD 68 CD68 CD68 antigen CD68 molecule CD68_HUMAN DKFZp686M18236 gp11 Gp110 LAMP4 Macrophage antigen CD68 (microsialin) MACROPHAGE ANTIGEN CD68 Macrosialin SCARD1 Scavenger receptor class D member 1

Images

Fig1: Western blot analysis of CD68 on different lysates with Mouse anti-CD68 antibody (EM1901-95) at 1/1,000 dilution.

Lane 1: A431 cell lysate
Lane 2: THP-1 cell lysate
Lane 3: U-937 cell lysate
Lane 4: Jurkat cell lysate (negative)

Lysates/proteins at 20 µg/Lane.

Predicted band size: 37 kDa
Observed band size: 100-150 kDa

Exposure time: 3 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM1901-95) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig2: Fluorescence multiplex immunohistochemical analysis of the human pancreatic carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD68 (EM1901-95, green), anti-CD163 (ET1704-43, red) and anti-PanCK (HA601094, violet) on human pancreatic carcinoma. Panel B: anti- CD68 stained on M1 macrophages. Panel C: anti-CD163 stained on M2 macrophages cells. Panel D: anti-panCK stained on cancer cells. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of EM1901-95 (1/3,000 dilution), ET1704-43 (1/3,000 dilution), and HA601094 (1/3,000 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Nikon ECLIPSE Ni-E microscope.

	Fig3: Fluorescence multiplex immunohistochemical analysis of human colon carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD68 (EM1901-95, Yellow) and anti-CD56 (ET1702-43, Red) on human colon carcinoma. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in two rounds of staining: in the order of EM1901-95 (1/3,000 dilution) and ET1702-43 (1/1,000 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Zeiss Observer 7 Inverted Fluorescence Microscope.
	Fig4: Fluorescence multiplex immunohistochemical analysis of human cervical carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD66b (HA500100, Green), anti-CD11b (ET1706-04, Red) and anti-CD68 (EM1901-95, Yellow) on human cervical carcinoma. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of HA500100 (1/1,000 dilution), ET1706-04 (1/1,000 dilution) and EM1901-95 (1/3,000 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Zeiss Observer 7 Inverted Fluorescence Microscope.
	Fig5: Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Rabbit anti-CD68 antibody (EM1901-95) at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (EM1901-95) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig6: Immunohistochemical analysis of paraffin-embedded human lung tissue using anti-CD68 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (EM1901-95, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig7: Flow cytometric analysis of THP-1 cells labeling CD68.

Cells were fixed and permeabilized. Then stained with the primary antibody (EM1901-95, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.