Lane 1: Wild type HAP1 whole cell lysate (20 µg)Lane 2: empty laneLane 3: KO HAP1 LMNB1 whole cell lysate (20 µg)Lane 4: empty laneLanes 1 - 4: Merged signal (red and green). Green - ab16048 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab16048 was shown to specificallyreact with LMNB1 (Lamin B1) in wild type HAP1 cells.No band was observed when LMNB1 (Lamin B1)knockout samples were used. Ab16048 LMNB1 (Lamin B1) and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 0.1 μg per mLand 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Human and mouse cells stained with ab16048 (1/500). The cells were fixed and permeabilized in 4% formaldehyde, 0.2% Tritonm X100 for 10 minutes at room temperature, then washed 3x in PBS.

A: HeLa cells + ab16048 (green)B: HeLa cells counterstained with DAPI (blue)C: 3T3 cells + ab16048 (green)D: 3T3 cells counterstained with DAPI (blue)

IHC image ofLamin B1 staining inHuman normal Liverformalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16048, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Lane 1: Wild-type HAP1 nuclear lysate (10 µg) Lane 2:Lamin B1 knockout HAP1 nuclear lysate (10 µg)

Lanes 1 and 2: Green signal from target- ab16048 observed at68 kDa. Red signal from loading control- ab10799 observed at 18 kDa.ab16048 was shown to specifically react with lamin B1 in wild-type HAP1 cells. No band was observed knockout samples were used. Wild-type and lamin B1 knockout samples were subjected to SDS-PAGE. ab16048 and ab10799 (loading control to histone H3 at 0.1µg/mL) were bothincubated overnight at 4°C. 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 1/10,000 dilution for 1 h at room temperature before imaging.

ICC/IF image of ab16048 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab16048, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

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ab16048 staining Lamin B1 in human infantile fibromatosis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% FBS/BSA for 3 hours at room temperature; antigen retrieval was by heat mediation in Tris pH9. Samples were incubated with primary antibody (1/100 in TBS + 1% BSA + 1% FBS) for 16 hours. An undilutedHRP-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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Human and mouse cells stained with ab16048 (1/500). The cells were fixed in 100% methanol for 6 minutes at -20°C, then washed once in PBS.

A: HeLa cells + ab16048 (green)B: HeLa cells counterstained with DAPI (blue)C: 3T3 cells + ab16048 (green)D: 3T3 cells counterstained with DAPI (blue)