Anti-COL18A1 antibody [EPR23481-21]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-COL18A1 antibody [EPR23481-21] (AB275746)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized U-937 (Human histiocytic lymphoma monocyte, Left) / HepG2 (Human hepatocellular carcinoma epithelial cell, Right) cells labelling COL18A1 with ab275746 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti-rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : U937 cell (PMID : 15451191).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COL18A1 antibody [EPR23481-21] (AB275746)

Immunohistochemical analysis of paraffin-embedded Human bladder cancer tissue labeling COL18A1 with ab275746 at 1/4000 (0.134 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on blood vessels of human bladder cancer (PMID : 21815140). The section was incubated with ab275746 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-COL18A1 antibody [EPR23481-21] (AB275746)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 cells labelling COL18A1 with ab275746 at 1/50 (8.26 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HepG2 cells. Negative control : U937 cell (PMID : 15451191). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COL18A1 antibody [EPR23481-21] (AB275746)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling COL18A1 with ab275746 at 1/4000 (0.134 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human kidney (PMID : 12588956). The section was incubated with ab275746 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.

• WB

Lab

Western blot - Anti-COL18A1 antibody [EPR23481-21] (AB275746)

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

The expression profile observed is consistent with what has been described in the literature (PMID : 11581192, 15451191).

Negative control : U937 (PMID : 15451191).

The lanes 1 of blot were developed using a higher sensitivity ECL substrate.

Exposure time : Lane 1 : 3 minutes Lane 2-3 : 70 seconds.

All lanes:

Western blot - Anti-COL18A1 antibody [EPR23481-21] (ab275746) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

Huh7 (human hepatocellar carcinoma epithelial cell), whole cell lysate at 40 µg

Lane 3:

U937 (human histiocytic lymphoma monocyte), whole cell lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 178 kDa

Observed band size: 178 kDa

false

• IP

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Immunoprecipitation - Anti-COL18A1 antibody [EPR23481-21] (AB275746)

COL18A1 was immunoprecipitated from 0.35 mg Huh7 (human hepatocellular carcinoma epithelial cell), whole cell lysate with ab275746 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275746 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Huh7 (human hepatocellular carcinoma epithelial cell), whole cell lysate 10 ug

Lane 2 : ab275746 IP in Huh7 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of 275746 in Huh7 whole cell lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-COL18A1 antibody [EPR23481-21] (ab275746)

Predicted band size: 178 kDa

Observed band size: 178 kDa

false