Anti-NPTXR + NPTX2 + NPTX1 抗体 [EPR25684-9] (ab289861)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling NPTXR+NPTX2+NPTX1 with ab289861 at 1/2000 dilution, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human cerebrum. The section was incubated with ab289861 for 30 mins at room temperature.

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

The immunostaining was performed on a Leica Biosystems BOND® RX instrumentCounter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum (fresh) tissue labelling NPTXR+NPTX2+NPTX1 with ab289861 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution (Green). Positive staining on mouse cerebrum (fresh). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution.

All lanes : Anti-NPTXR + NPTX2 + NPTX1 antibody [EPR25684-9] (ab289861) at 1/1000 dilution

Lane 1 : IMR-32 (Human neuroblastoma neuroblast) whole cell lysate at 20 µg
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 3 : Mouse brain tissue lysate at 20 µg
Lane 4 : Mouse skeletal muscle tissue lysate at 40 µg
Lane 5 : Mouse spleen tissue lysate at 20 µg
Lane 6 : Rat brain tissue lysate at 20 µg
Lane 7 : Rat skeletal muscle tissue lysate at 40 µg
Lane 8 : Rat spleen tissue lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 52 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

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

Eposure times: 

Lane1-2: 180 seconds;
Lane3-8: 70 seconds. 

Samples are non-boiled as boiling may cause protein aggregates.
The observed MW are consistent with what has been described in the literature (PMID: 32847597, PMID: 32273328, PMID: 28440221)
Low expression: Hela, skeletal muscle, spleen (PMID: 32847597, PMID: 32273328, PMID: 28440221)
The bands nearby 40 kDa could be non-specific bands. 

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling NPTXR+NPTX2+NPTX1 with ab289861 at 1/2000 dilution, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on mouse cerebrum. The section was incubated with ab289861 for 30 mins at room temperature.

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

The immunostaining was performed on a Leica Biosystems BOND® RX instrumentCounter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

All lanes : Anti-NPTXR + NPTX2 + NPTX1 antibody [EPR25684-9] (ab289861) at 1/1000 dilution

Lane 1 : His-tagged human NPTXR recombinant protein fragment 10ng
Lane 2 : His-tagged human NPTX1 full length recombinant protein (without signal peptide) 10ng
Lane 3 : Human NPTX2 full length recombinant protein (without signal peptide) 10ng

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?

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

Eposure times: 5.5 seconds

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling NPTXR+NPTX2+NPTX1 with ab289861 at 1/2000 dilution, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on rat cerebrum. The section was incubated with ab289861 for 30 mins at room temperature.

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

The immunostaining was performed on a Leica Biosystems BOND® RX instrumentCounter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse skeletal muscle (fresh) tissue labelling NPTXR+NPTX2+NPTX1 with ab289861 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution (Green). Negative control (PMID: 9261167). No staining on mouse skeletal muscle (fresh). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution. 

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling NPTXR+NPTX2+NPTX1 with ab289861 at 1/2000 dilution, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Negative control: no staining on human liver. The section was incubated with ab289861 for 30 mins at room temperature. 

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

The immunostaining was performed on a Leica Biosystems BOND® RX instrumentCounter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum (fresh) tissue labelling NPTXR+NPTX2+NPTX1 with ab289861 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution (Green). Positive staining on rat cerebrum. The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution. 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat skeletal muscle (fresh) tissue labelling NPTXR+NPTX2+NPTX1 with ab289861 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution (Green). Negative control (PMID: 9261167). No staining on rat skeletal muscle (fresh). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution.