Anti-CSF-1-R antibody [EPR28407-22]

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-CSF-1-R antibody [EPR28407-22] (AB313648)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human tonsil tissue staining CARD9 with ab322105 at a 1/100 (4.99 µg/ml) dilution, ab313648 anti-CSF-1-R used at 1/100 (5.26 µg/ml) dilution.

Panel A : merged staining of anti-CARD9 (green; Opal™520) and anti-CSF-1-R (magenta; Opal™570) on human tonsil.

Panel B : anti-CARD9 staining immune cells in human tonsil.

Panel C : anti-CSF-1-R staining macrophages and monocytes in human tonsil.

Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab322105 and ab313648 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSF-1-R antibody [EPR28407-22] (AB313648)

Immunohistochemical analysis of paraffin-embedded Human colorectal car tissue labeling MCSF Receptor with ab313648 at 1/100 (5.26 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on immune cells of human colorectal carcinoma.The section was incubated with ab313648 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSF-1-R antibody [EPR28407-22] (AB313648)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling MCSF Receptor with ab313648 at 1/100 (5.26 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on immune cells of human colon.The section was incubated with ab313648 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSF-1-R antibody [EPR28407-22] (AB313648)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling MCSF Receptor with ab313648 at 1/100 (5.26 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human tonsil (PMID : 26066800)The section was incubated with ab313648 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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CSF-1-R antibody [EPR28407-22] (AB313648)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Tween-20 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling MCSF Receptor with ab313648 at 1/50 (10.52 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 and membranous staining in THP-1 cell line.Negative control : Raji.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 (Red). 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.

• IP

Lab

Immunoprecipitation - Anti-CSF-1-R antibody [EPR28407-22] (AB313648)

MCSF Receptor was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab313648 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed from the immunoprecipitate using ab313648 at 1/1000 dilution. Secondary antibody details (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1 : THP-1 (human monocytic leukemia monocyte) whole cell lysate 20 μg.

Lane 2 : ab313648 IP in THP-1 (human monocytic leukemia monocyte) whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab313648 in THP-1 whole cell lysate.

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

Exposure time : 180.

Lysates were freshly made and used for immunoprecipitation immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-CSF-1-R antibody [EPR28407-22] (ab313648) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

ab313648 IP in THP-1 (human monocytic leukemia monocyte) whole cell lysate.

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 108 kDa

Observed band size: 180 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CSF-1-R antibody [EPR28407-22] (AB313648)

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

Negative control : Raji, HDLM-2 (PMID : 26066800).

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 14673177).

In lanes 1-3, the lysates were stored at -80°C prior to Western Blotting. The bands beneath the target band (140 kDa) are likey to be degradation products. In lanes 4-5, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CSF-1-R antibody [EPR28407-22] (ab313648) at 1/1000 dilution

Lanes 1 and 4:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lanes 2 and 5:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 3:

HDLM-2 (human Hodgkin lymphoma) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 190 kDa,140 kDa

true

Exposure time: 180s