Recombinant Anti-P2Y12 antibody [EPR23511-72] (ab254347)

Fluorescence multiplex immunohistochemical analysis of the human cerebellum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (ab254363, green; Opal™520) and anti-P2Y12 (ab254347 red; Opal™570) on human cerebellum. Panel B: anti-P2Y12 stained on microglial cells. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), ab254363 (1/2000 dilution), and ab254347 (1/1000 dilution) for 30 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, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

Fluorescence multiplex immunohistochemical analysis of the human cerebrum (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-GFAP (ab68428, gray; Opal™690), anti-Myelin PLP (ab254363, green; Opal™520) and anti-P2Y12 (ab254347, red; Opal™570) on human cerebrum. Panel B: anti-P2Y12 stained on microglial cells. Panel C: anti-Myelin PLP stained on myelin sheaths of oligodendrocytes. Panel D: anti-GFAP stained on astrocytes. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab68428 (1/50 dilution), ab254363 (1/2000 dilution), and ab254347 (1/1000 dilution) for 30 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, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

Immunohistochemical analysis of paraffin-embedded Human glioma tissue labeling P2Y12 with ab254347 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human glioma (PMID: 30832693). The section was incubated with ab254347 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Dot blot analysis of P2Y12 using ab254347 at 1/1000 dilution (0.569 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Lane 1: Human P2Y12 immunogen peptide 

Lane 2: Human P2Y12 non-immunogen peptide

Lane 3: Human P2Y12 non-immunogen peptide

Lane 4: BSA

Exposure time: 3 minutes

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Fluorescence multiplex immunohistochemical analysis of the Human cerebrum (Formalin/PFA-fixed paraffin-embedded sections).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Opal Polymer HRP Ms + Rb was used as a secondary antibody.DAPI (blue) was used as a nuclear counter stain.

Panel A: Merged staining of anti-GFAP (gray; Opal™690), anti-P2Y12 (green; Opal™520) and anti-MAP2 (red; Opal™570) on human cerebrum.
Panel B: Anti-MAP2 stained cell body and dendrites of neurons.
Panel C: Anti-P2Y12 stained on microglial cells.
Panel D: Anti-GFAP stained on astrocytes.

The section was incubated in three rounds of staining: in the order of ab68428, ab254347, and ab254263 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Fluorescence multiplex immunohistochemical analysis of the Human cerebellum (Formalin/PFA-fixed paraffin-embedded sections).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
Opal Polymer HRP Ms + Rb was used as a secondary antibody.DAPI (blue) was used as a nuclear counter stain.

Panel A: Merged staining of anti-GFAP (gray; Opal™690), anti-P2Y12 (green; Opal™520) and anti-MAP2 (red; Opal™570) on human cerebellum.
Panel B: Anti-MAP2 stained cell body and dendrites of neurons.
Panel C: Anti-P2Y12 stained on microglial cells.
Panel D: Anti-GFAP stained on astrocytes.

The section was incubated in three rounds of staining: in the order of ab68428, ab254347, and ab254263 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling P2Y12 with ab254347 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on microglia (PMID: 30832693). The section was incubated with ab254347 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.