Cancer Associated Fibroblast Marker Antibody Kit

Western blot analysis of Vimentin on different lysates with Rabbit anti-Vimentin antibody (ET1610-39) at 1/20,000 dilution.

Lane 1: HeLa cell lysate (10 µg/Lane)
Lane 2: HEK-293 cell lysate (10 µg/Lane)
Lane 3: Jurkat cell lysate (10 µg/Lane)
Lane 4: NIH/3T3 cell lysate (10 µg/Lane)
Lane 5: C6 cell lysate (10 µg/Lane)
Lane 6: C2C12 cell lysate (10 µg/Lane)
Lane 7: RAW264.7 cell lysate (10 µg/Lane)

Predicted band size: 54 kDa
Observed band size: 54 kDa

Exposure time: Lane 1-5: 3 seconds; Lane 6-7: 14 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 (ET1610-39) at 1/20,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.

Fluorescence multiplex immunohistochemical analysis of the human pancreatic carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD31 (M1511-8, green), anti-α-SMA (ET1607-53, red) and anti-FAP (ET1704-23, yellow) on human pancreatic carcinoma. Panel B: anti- CD31 stained on the endothelial cells. Panel C: anti-α-SMA stained on cancer-associated fibroblasts and smooth muscle cells. Panel D: anti-FAP stained on the cancer-associated fibroblasts. 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 M1511-8 (1/5000 dilution), ET1704-23 (1/1000 dilution), and ET1607-53 (1/3000 dilution) for 20 mins at room temperature. 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.

Western blot analysis of alpha smooth muscle Actin on different lysates with Rabbit anti-alpha smooth muscle Actin antibody (ET1607-53) at 1/20,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: Saos-2 cell lysate (15 µg/Lane)
Lane 2: A431 cell lysate (15 µg/Lane)
Lane 3: NIH/3T3 cell lysate (15 µg/Lane)
Lane 4: C2C12 cell lysate (15 µg/Lane)
Lane 5: Neuro-2a cell lysate (15 µg/Lane)
Lane 6: Mouse skin tissue lysate (30 µg/Lane)
Lane 7: Rat skin tissue lysate (30 µg/Lane)

Predicted band size: 42 kDa
Observed band size: 42 kDa

Exposure time: 46 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 (ET1607-53) at 1/20,000 dilution and competitor's antibody at 1/2,000 dilution were 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.

Immunofluorescence analysis of paraffin-embedded human kidney tissue labeling alpha smooth muscle Actin (ET1607-53).

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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibody alpha smooth muscle Actin (ET1607-53, red) at 1/1,000 dilution overnight at 4 ℃, washed with PBS.

iFluor™ 647 conjugate-Goat anti-Rabbit IgG (HA1123) was used as the secondary antibody at 1/1,000 dilution. DAPI was used as nuclear counterstain.

Fluorescence multiplex immunohistochemical analysis of the human pancreatic carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD31 (M1511-8, green), anti-α-SMA (ET1607-53, red) and anti-FAP (ET1704-23, yellow) on human pancreatic carcinoma. Panel B: anti- CD31 stained on the endothelial cells. Panel C: anti-α-SMA stained on cancer-associated fibroblasts and smooth muscle cells. Panel D: anti-FAP stained on the cancer-associated fibroblasts. 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 M1511-8 (1/5000 dilution), ET1704-23 (1/1000 dilution), and ET1607-53 (1/3000 dilution) for 20 mins at room temperature. 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.

Immunofluorescence analysis of paraffin-embedded human stomach cancer tissue labeling alpha smooth muscle Actin (ET1607-53) and ALDH2 (M1509-1).

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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibody alpha smooth muscle Actin (ET1607-53, red) at 1/1,000 dilution and ALDH2 (M1509-1, green) at 1/100 dilution overnight at 4 ℃, washed with PBS. iFluor™ 488 conjugate-Goat anti-Mouse IgG (HA1125) and iFluor™ 647 conjugate-Goat anti-Rabbit IgG (HA1123) was used as the secondary antibody at 1/1,000 dilution. DAPI was used as nuclear counterstain.

Fluorescence multiplex immunohistochemical analysis of Human non-small cell lung cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-PD-L1 (HA721176, red), anti-CD34 (ET1606-11, green), anti-Pan-CK (HA601138, cyan), anti-CD20 (HA721138, magenta), anti-αSMA (ET1607-53, yellow) and anti-CD57 (HA601114, white) on NSCLC. Panel B: anti-PD-L1 stained on dendritic cells and macrophages cells. Panel C: anti- CD34 stained on endothelial cells. Panel D: anti-Pan-CK stained on cancer cells. Panel E: CD20 stained on B cells. Panel F: anti-αSMA stained on cancer-associated fibroblasts and smooth muscle cells. Panel G: anti-CD57 stained on NK cells and T 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 six rounds of staining: in the order of HA721176 (1/1,000 dilution), ET1606-11 (1/1,000 dilution), HA601138 (1/3,000 dilution), HA721138 (1/2,000 dilution), ET1607-53 (1/3,000 dilution) and HA601114 (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 Olympus VS200 Slide Scanner.

Fluorescence multiplex immunohistochemical analysis of mouse liver (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-Albumin (ET1702-55, Violet), anti-SOX9 (ET1611-56, Yellow) and anti-αSMA (ET1607-53, White) on liver. 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 ET1702-55 (1/3,000 dilution), ET1611-56 (1/1,500 dilution) and ET1607-53 (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.

Fluorescence multiplex immunohistochemical analysis of the human gastric cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD31 (M1511-8, red), anti-αSMA (ET1607-53, gray), anti-CD11b (ET1706-04, cyan), anti-panCK (HA601138, magenta) and anti-CD3 (HA720082, yellow) on human gastric cancer. Panel B: anti- CD31 stained on the endothelial cells. Panel C: anti-αSMA stained on cancer-associated fibroblasts and smooth muscle cells. Panel D: anti-CD11b stained on myeloid cells. Panel E: anti-panCK stained on cancer cells. Panel F: anti-CD3 stained on T 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 M1511-8 (1/1,000 dilution), ET1607-53 (1/2,000 dilution), ET1706-04 (1/1,000 dilution), HA601138 (1/3,000 dilution), and HA720082 (1/500 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 mouse lung (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-TTF1 (HA720067, Red), anti-RAGE (ET1702-27, Green), anti-aSMA (ET1607-53, Cyan) and anti-Ki67 (HA721115, Yellow) on mouse lung. 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 four rounds of staining: in the order of HA720067 (1/4,000 dilution), ET1702-27 (1/3,000 dilution), ET1607-53 (1/10,000 dilution) and HA721115 (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 mouse kidney (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-NPHS2 (ET7107-34, Red), anti-AQP1 (ET1703-34, Green), anti-Laminin beta 1 (ET1703-14, Cyan) and anti-aSMA (ET1607-53, Magenta) on kidney. 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 four rounds of staining: in the order of ET7107-34 (1/1,000 dilution), ET1703-34 (1/5,000 dilution), ET1703-14 (1/1,000 dilution) and ET1607-53 (1/10,000 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.