CD20 Recombinant Rabbit Monoclonal Antibody [PD00-02]

Fluorescence multiplex immunohistochemical analysis of Tertiary Lymphoid Structures in Human Small Cell Lung Cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, green), anti-PD-L1 (HA721176, cyan), anti-CD56 (ET1702-43, magenta) and anti-CD3 (HA720082, yellow) on tertiary lymphoid structures. Panel B: anti- CD20 stained on B cells. Panel C: anti-PD-L1 stained on dendritic cells and macrophages cells. Panel D: anti-CD56 stained on NKT cells. Panel E: 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 four rounds of staining: in the order of HA721138 (1/1,500 dilution), HA721176 (1/1,000 dilution), ET1702-43 (1/1,000 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 the human non-small cell lung cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, green), anti-CD68 (HA601115, gray), anti-PD-L1 (HA721176, cyan), anti-panCK (HA601138, magenta) and anti-CD3 (HA720082, yellow) on human non-small cell lung cancer. Panel B: anti- CD20 stained on B cells. Panel C: anti-CD68 stained on macrophage M1 and macrophage M2. Panel D: anti-PD-L1 stained on dendritic cells and macrophages 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 HA721138 (1/1,500 dilution), HA601115 (1/2,000 dilution), HA721176 (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 tertiary lymphoid structures in human prostate cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, green), anti-CD21 (HA721163, cyan) and anti-CD4 (ET1609-52, yellow) on tertiary lymphoid structures. Panel B: anti- CD20 stained on B cells. Panel C: anti-CD21 stained on naive B-cell, memory B-cell and plasma cells. Panel D: anti-CD4 stained on helper T cells and Treg 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 HA721138 (1/1,500 dilution), HA721163 (1/1,000 dilution), and ET1609-52 (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 Human tonsil (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-BCL6 (HA601083, Red), anti-HLA-DPB1 (ET1704-13, Green), anti-Tryptase (ET1610-64, White), anti-CD20 (HA721138, Magenta) and anti-CD45 (ET7111-03, 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 five rounds of staining: in the order of HA601083 (1/200 dilution), ET1704-13 (1/2,000 dilution), ET1610-64 (1/5,000 dilution), HA721138 (1/2,000 dilution) and ET7111-03 (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 Human tonsil (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD68 (HA601115, Red), anti-CD38 (HA721268, Green), anti-CD23 (HA721139, White), anti-CD11C (ET1606-19, Cyan), anti-CD45 (ET7111-03, Magenta) and anti-CD20 (HA721138, Yellow) on tonsil. Panel B: anti-CD68 stained on Macrophage. Panel C: anti-CD38 stained on lymphocyte subsets. Panel D: anti-CD11C stained on dendritic cells. Panel E: CD45 stained on lymphocytes. Panel F: anti-CD20 stained on B cells. Panel G: anti-CD23 stained on follicular dendritic 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 HA601115 (1/2,000 dilution), HA721268 (1/1,000 dilution), ET1606-19 (1/1,000 dilution), ET7111-03 (1/500 dilution), HA721138 (1/2,000 dilution) and HA721139 (1/800 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 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 human tonsil (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, Cyan), anti-CD38 (HA721268, Violet) and anti-CD57 (HA601114, 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 HA721138 (1/2,000 dilution), HA721268 (1/1,000 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 Zeiss Observer 7 Inverted Fluorescence Microscope.

☑ Relative expression (RE)

Western blot analysis of CD20 on different lysates with Rabbit anti-CD20 antibody (HA721138) at 1/2,000 dilution.

Lane 1: Raji cell lysate
Lane 2: 293T cell lysate (negative)
Lane 3: Ramos cell lysate
Lane 4: HeLa cell lysate (negative)
Lane 5: Daudi cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 33 kDa
Observed band size: 33 kDa

Exposure time: 4 seconds; ECL: K1801;

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 (HA721138) at 1/2,000 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.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue with Rabbit anti-CD20 antibody (HA721138) at 1/2,000 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 (HA721138) at 1/2,000 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.

Immunohistochemical analysis of paraffin-embedded human lymph nodes tissue with Rabbit anti-CD20 antibody (HA721138) at 1/2,000 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 (HA721138) at 1/2,000 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.

Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-CD20 antibody (HA721138) 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 (HA721138) 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.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue with Rabbit anti-CD20 antibody (HA721138) 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 (HA721138) 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.

☑ Relative expression (RE)

Immunocytochemistry analysis of Raji (positive) and 293T (negative) labeling CD20 with Rabbit anti-CD20 antibody (HA721138) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-CD20 antibody (HA721138) at 1/500 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 (HA601187, 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.

CD20 was immunoprecipitated from 0.2 mg Raji cell lysate with HA721138 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA721138 at 1/2,000 dilution. Mouse anti Rabbit IgG heavy chain (Fc) secondary antibody (M1003-7) at 1/10,000 dilution was used for 1 hour at room temperature.

Lane 1: Raji cell lysate (input)
Lane 2: HA721138 IP in Raji cell lysate
Lane 3: Rabbit IgG instead of HA721138 in Raji cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 59 seconds; ECL: K1801