Recombinant Anti-Lamin B1 antibody [EPR22165-121] (ab229025)

Lanes 1- 2: Merged signal (red and green). Green - ab229025 observed at 66-70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab229025 was shown to react with Lamin B1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255404 (knockout cell lysate ab263825) was used. Wild-type HeLa and LMNB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab229025 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Multiplex immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue.

Panel A:  Merged staining of Collagen VI (ab182744; green), anti-CD68 (ab213363; red) and anti-Lamin B1 (ab229025; magenta).

Panel B: Anti-Collagen VI (green) stained on extracellular matrix.

Panel C: Anti-CD68 (red) stained on Kupffer cells.

Panel D: Anti-Lamin B1 (magenta) stained on nuclear envelope.

Key protocol steps:  The section was incubated in three rounds of staining with ab182744 (1/1000 dilution), ab213363 (1/1000 dilution) and ab229025 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by tyramide signal amplification with the appropriate fluorophore. Heat mediated antigen retrieval was used (Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins after every round of antibody/fluorophore staining.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

DAPI was used as a nuclear counter stain. A ready-to-use anti-Rabbit and Mouse Polymer HRP was used as a secondary.

ab229025 was shown to specifically react with Lamin B1 in wild-type HAP1 cells as signal was lost in Lamin B1 knockout cells. Wild-type and Lamin B1 knockout samples were subjected to SDS-PAGE. ab229025 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Lamin B1 (Green) with ab229025 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing nuclear membranous staining in HeLa cells. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as a counterstain at 1/200 dilution. The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on mouse liver (PMID: 19925772) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling Lamin B1 (Green) with ab229025 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution. Confocal image showing nuclear membranous staining in NIH/3T3 cells. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as a counterstain at 1/200 dilution. The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

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

Exposure time:

Lane 1:3 seconds;

Lanes 2,3,4 and 5:10 seconds.

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

Exposure time:

Lane 1:10 seconds; Lanes 2 and 3: 59 seconds; Lane 4:3 seconds; Lanes 5,6 and 7:6 seconds.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on rat cerebrum (PMID: 26469707, PMID: 19925772) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human breast tissue stained for Lamin B1 using ab229025 at 1/2000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear envelope staining on human breast (PMID: 26469707) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

Lamin B1 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab229025 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229025 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg (input).
Lane 2: ab229025 IP in HeLa whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229025 in HeLa whole cell lysate.

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

A very weak band around 66 kDa detected in lane 3 is due to spill over from lane2 (+).

Lamin B1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate with ab229025 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229025 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10 μg (input).
Lane 2: ab229025 IP in NIH/3T3 whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229025 in NIH/3T3 whole cell lysate.

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

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cell line labeling Lamin B1 with ab229025 at 1/500 dilution (Red) compared with aRabbit monoclonal IgGisotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^� 488, ab150077) was used as the secondary antibody at 1/2000 dilution. 

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Lamin B1 with ab229025 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgGisotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^� 488, ab150077) was used as the secondary antibody at 1/2000 dilution.