Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling MARCO with ab239369 at 1/500 dilution (1.11 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on Kupffer cells of mouse liver is observed. The section was incubated with ab239369 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 solution 2) for 20mins.

All lanes : Anti-MARCO antibody [EPR22944-64] (ab239369) at 1/1000 dilution

Lane 1 : Untreated J774A.1 (mouse reticulum cell sarcoma monocyte macrophage), whole cell lysate
Lane 2 : J774A.1 treated with 1ug/ml Lipopolysaccharides (LPS) for 24 hours, whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Observed band size: 55-75 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST .

Exposure time: 3 seconds.

The expression of MARCO/Macrophage receptor with collagenous structure is induced by Lipopolysaccharides (LPS) treatment (PMID: 9916718).

All lanes : Anti-MARCO antibody [EPR22944-64] (ab239369) at 1/1000 dilution

Lane 1 : Mouse spleen lysate
Lane 2 : Mouse spleen lysate treated with Peptide:N-glycosidase F (PNGase F)

Lysates/proteins at 20 µg per lane.

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

Observed band size: 53,75 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

75 kDa (glycosylated from), 53 kDa (deglycosylated form).

Blocking and Dilution Buffer and concentration: 5% NFDM/TBST.

N-glycosylation is crucial for MARCO/Macrophage receptor with collagenous structure intercellular trafficking and surface targeting (PMID: 26892079).

MARCO was immunoprecipitated from 0.35 mg mouse spleen lysate 10µg with ab239369 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab239369 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse spleen lysate 10µg.

Lane 2: ab239369 IP in mouse spleen lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab239369 in mouse spleen lysate.

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

Exposure time: 8 seconds.

 

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling MARCO with ab239369 at 1/500 dilution (1.11 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on macrophages of mouse spleen (PMID: 12874264, 15494482) is observed. The section was incubated with ab239369 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® 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 solution 2) for 20mins.

4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen section of mouse spleen tissue labeling MARCO using ab239369 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (green). The nuclear counterstain is DAPI (blue). Positive staining on mouse splenic marginal zone (PMID: 24670793) is observed.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized J744A.1 (Mouse reticulum cell sarcoma monocyte macrophage) cells labeling MARCO with ab239369 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in J744A.1 cells treated with LPS (1 μg/ml) for 24h. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.