Product namePD-L1 / PD1 Multiplex IHC-IF Antibody Panel (PD-L1, PD1, CD68, CD3, Ki67, panCK)
Species reactivityReacts with: Human
PD-L1 / PD1 Multiplex IHC-IF Antibody Panel (PD-L1, PD1, CD68, CD3, Ki67, panCK) is specifically designed and validated for multiplex analysis of the tumor microenvironment (TME) using tyramide signal amplification (TSA) on human formalin fixed paraffin-embedded tissue (FFPE) sections. The order of staining and antibody dilutions have been optimized on human tonsil FFPE tissue.
This panel empowers researchers to explore the spatial interactions of these important immuno-oncology biomarkers, immune checkpoint proteins and immune cell-phenotyping markers within the tumor micro-environment. It allows for the 6 targets to be fluorescently detected on the same tissue section, plus a nuclear counterstain (DAPI).
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT, Akoya Biosciences®). Please refer to this kit’s protocol for directions on how to perform the immunostaining.
The panel contains enough reagents for a minimum of 50 slides.
Panel contents, recommended order of staining and dilutions:
1. Recombinant PD1 antibody [CAL20] ab237728 (50 µl)
1/500 dilution; Opal™520
2. Recombinant PD-L1 antibody [CAL10] ab237726 (50 µl)
1/500 dilution; Opal™540
3. Recombinant CD68 antibody [SP251] ab192847 - Macrophage marker (50 µl)
1/300 dilution; Opal™570
4. CD3 antibody [SP7] ab16669 - T cell marker (50 µl)
1/300 dilution; Opal™620
5. Recombinant Ki67 antibody [SP6] ab16667 - Proliferation marker (50 µl)
1/200 dilution; Opal™650
6. Pan-cytokeratin antibody [C-11] ab7753 - Epithelial tissue marker (50 µg)
1/200 dilution; Opal™690
Tested applicationsSuitable for: mIHCmore details
Storage instructionsPlease refer to protocols.
Components 1 kit ab16669 - Anti-CD3 antibody [SP7] 1 x 50µl ab7753 - Anti-pan Cytokeratin antibody [C-11] 1 x 50µg ab192847 - Recombinant Anti-CD68 antibody [SP251] 1 x 50µl ab16667 - Recombinant Anti-Ki67 antibody [SP6] 1 x 50µl ab237728 - Recombinant Anti-PD1 antibody [CAL20] 1 x 50µl ab237726 - Recombinant Anti-PD-L1 antibody [CAL10] 1 x 50µl
Cellular localizationCD3: Membrane. CD68: Cell membrane and Endosome membrane. Lysosome membrane. Ki67: Chromosome. Nucleus. Nucleus, nucleolus. Associates with the surface of the mitotic chromosome, the perichromosomal layer, and covers a substantial fraction of the mitotic chromosome surface (PubMed:27362226). Associates with satellite DNA in G1 phase (PubMed:9510506). Binds tightly to chromatin in interphase, chromatin-binding decreases in mitosis when it associates with the surface of the condensed chromosomes (PubMed:15896774, PubMed:22002106). Predominantly localized in the G1 phase in the perinucleolar region, in the later phases it is also detected throughout the nuclear interior, being predominantly localized in the nuclear matrix (PubMed:22002106). pan Cytokeratin: Cytoplasmic PD1: Membrane. PD-L1: Cell membrane and Endomembrane system.
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab269812 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use at an assay dependent concentration.
Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).
Merged staining of anti-PD1 (ab237728; orange; Opal™520), anti-PDL1 (ab237726; green; Opal™540), anti-CD68 (ab192847; yellow; Opal™570), anti-CD3 (ab16669; red; Opal™620), anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (ab7753; grey; Opal™690).
The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT, Akoya Biosciences®).
The section was incubated in six rounds of staining; in the order of ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.
Sodium citrate antigen retrieval (Leica ER1, pH6.0, 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.
DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab269812 has not yet been referenced specifically in any publications.