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☑ Cell treatment (CT)
Western blot analysis of PD-L1 on different lysates with Rabbit anti-PD-L1 antibody (HA722184) at 1/2,000 dilution.
Lane 1: RAW264.7 cell lysate
Lane 2: RAW264.7 treated with 10μg/mL LPS for 8 hours cell lysate
Lane 3: J774A.1 cell lysate
Lane 4: J774A.1 treated with 1μg/mL LPS for 24 hours cell lysate
Lane 5: Mouse spleen tissue lysate
Lysates/proteins at 30 µg/Lane.
Predicted band size: 33 kDa
Observed band size: 45-60 kDa
Exposure time: 46 seconds; ECL: K1802;
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 (HA722184) 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.
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Fluorescence multiplex immunohistochemical analysis of mouse spleen (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-PD-L1 (HA722184, Green) and anti-Ki67 (HA721115, Violet) on spleen. 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 HA722184 (1/500 dilution) and HA721115 (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.
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Immunohistochemical analysis of paraffin-embedded mouse thymus tissue with Rabbit anti-PD-L1 antibody (HA722184) 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 (HA722184) 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.
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Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-PD-L1 antibody (HA722184) 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 (HA722184) 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.
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