CD14 Recombinant Rabbit Monoclonal Antibody [SC69-02]

Western blot analysis of CD14 on different lysates with Rabbit anti-CD14 antibody (ET1610-85) at 1/5,000 dilution.

Lane 1: THP-1 cell lysate
Lane 2: RAW264.7 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 40 kDa
Observed band size: 60 kDa

Exposure time: 1 minute 40 seconds;
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 (ET1610-85) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of CD14 on different lysates with Rabbit anti-CD14 antibody (ET1610-85) at 1/1,000 dilution.

Lane 1: SW480-si NT cell lysate
Lane 2: SW480-si CD14 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 40 kDa
Observed band size: 50-60 kDa

Exposure time: 7 seconds; ECL: merk
4-20% SDS-PAGE gel.

ET1610-85 was shown to specifically react with CD14 in SW480-si NT cells. Weakened band was observed when SW480-si CD14 sample was tested. SW480-si NT and SW480-si CD14 samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1610-85, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

Fluorescence multiplex immunohistochemical analysis of Human tonsil (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD14 (ET1610-85, Red), anti-CD4 (ET1609-52, Green), anti-CD57 (HA601114, White), anti-CD15 (HA721246, Cyan)and anti-Tryptase (ET1610-64, Magenta) on tonsil. Panel B: anti- CD14 stained on monocytes. Panel C: anti-CD4 stained on helper T cells and Treg cells. Panel D: anti-CD57 stained on NK cells and T cells. Panel E: CD15 stained on granulocytes and monocytes. Panel F: anti-Tryptase stained on Mast 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 five rounds of staining: in the order of ET1610-85 (1/800 dilution), ET1609-52 (1/800 dilution), HA601114 (1/1,000 dilution), HA721246 (1/500 dilution), and ET1610-64 (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 Olympus VS200 Slide Scanner.

Fluorescence multiplex immunohistochemical analysis of the human cervical cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD14 (ET1610-85, red), anti-S100A9 (ET1702-73, green), anti-CD68 (HA601115, cyan), anti-panCK (HA601138, magenta) and anti-CD163 (ET1704-43, yellow) on human cervical cancer. Panel B: anti- CD14 stained on monocyte and MDSCs. Panel C: anti-S100A9 stained on MDSCs. Panel D: anti-CD68 stained on macrophage M1 and macrophage M2. Panel E: anti-panCK stained on tumor cells. Panel F: anti-CD163 stained on macrophage M2. 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 five rounds of staining: in the order of ET1610-85 (1/1,000 dilution), ET1702-73 (1/1,000 dilution), HA601115 (1/2,000 dilution), HA601138 (1/3,000 dilution), and ET1704-43 (1/2,000 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 Olympus VS200 Slide Scanner.

Fluorescence multiplex immunohistochemical analysis of human tonsil (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD14 (ET1610-85, Green), anti-CD21 (HA721163, Red) and anti-Granzyme B (HA500252, Yellow) on tonsil. 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 ET1610-85 (1/800 dilution), HA721163 (1/1,000 dilution) and HA500252 (1/200 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.

Immunocytochemistry analysis of THP-1 cells labeling CD14 with Rabbit anti-CD14 antibody (ET1610-85) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CD14 antibody (ET1610-85) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunohistochemical analysis of paraffin-embedded human lymph nodes tissue with Rabbit anti-CD14 antibody (ET1610-85) at 1/800 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 (ET1610-85) at 1/800 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.

Immunocytochemistry analysis of NIH/3T3 cells labeling CD14 with Rabbit anti-CD14 antibody (ET1610-85) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CD14 antibody (ET1610-85) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-CD14 antibody (ET1610-85) at 1/800 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 (ET1610-85) at 1/800 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.

Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-CD14 antibody (ET1610-85) at 1/200 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 (ET1610-85) at 1/200 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.

Flow cytometric analysis of human peripheral blood cells labelling CD14 (ET1610-85).