重组Anti-Lrp2 / Megalin抗体[RM1150] (ab317333)

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

Negative control: HeLa, Raji (PMID: 35839778), human skeletal muscle.

In Western blot, Anti-Vinculin antibody [EPR8185]  (ab129002) staining at 1/10000 dilution.

Exposure time: Lanes 1-4, 6-7: 10 seconds; Lanes 5: 1 second;

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Lrp2 / Megalin with ab317333 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human kidney. The section was incubated with ab317333 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Lrp2 / Megalin with ab317333 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human placenta. The section was incubated with ab317333 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human clear cell renal cell carcinoma tissue labeling Lrp2 / Megalin with ab317333 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human clear cell renal cell carcinoma. The section was incubated with ab317333 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling Lrp2 / Megalin with ab317333 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control: no staining on human skeletal muscle. The section was incubated with ab317333 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized JEG-3 (human placenta epithelial cell) cells labelling Lrp2 / Megalin with ab317333 at 1/50 (10.0 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in JEG-3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control: HeLa (PMID: 36064583).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Flow cytometric analysis of HeLa (human cervical adenocarcinoma epithelial cell, Left) / JEG-3 (human placenta epithelial cell, Right) cells labelling Lrp2 / Megalin with ab317333 at 1/500 dilution (0.1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control: HeLa.
Gated on viable cell.

Lrp2 / Megalin was immunoprecipitated from 0.35 mg JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate with ab317333 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317333 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate
Lane 2: ab317333 IP in JEG-3 (Human placenta epithelial choriocarcinoma) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317333 in JEG-3 whole cell lysate

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

Exposure time: 8 seconds.