Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72]

• Flow Cyt

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Flow Cytometry - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

Flow cytometric analysis of unfixed non-permeabilized THP-1 (human monocytic leukemia cell line) cells treated with 10 ng/ml PMA for 18 hours, then serum starved for 8 hours, then 10 ng/ml PMA for 6 hours and add 1000 U IL4 for 2 hours, then add 10% serum for another 22 hours. DC-SIGN+DC-SIGNR was stained in treated (red) and untreated (green) cells using ab245115 at 1/600 dilution as compared to a Rabbit monoclonal IgG (ab172730, black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody / blue). The secondary antibody was a Goat anti rabbit IgG (Dylight^® 488, ab98462) used at 1/2000 dilution. Gated on viable cells. DC-SIGN expression is induced by PMA plus IL4 in THP-1 (PMID : 15070901; PMID : 22675249). DC-SIGNR/CD299 expression is induced by PMA in THP-1 (PMID 30077333).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling DC-SIGN + DC-SIGNR with ab245115 at 1/100 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Cytoplasmic staining on sinusoidal endothelial cells of human liver (PMID : 16816373) is observed. Counterstained with hematoxylin.

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

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia cell line) cells labeling DC-SIGN + DC-SIGNR with ab245115 at 1/100 dilution, followed by a AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) seconday antibody (green) at 1/1000 dilution.

Confocal image showing cytoplasmic and membranous staining in THP-1 cells treated with 10 ng/ml PMA for 18 hours, then serum starved for 8 hours, then 10 ng/ml PMA for 6 hours and add 1000 U IL4 for 2 hours, then add 10% serum for another 22 hours. DC-SIGN expression is induced by PMA plus IL4 in THP-1(PMID : 15070901; PMID : 22675249). DC-SIGNR/CD299 expression is induced by PMA in THP-1 (PMID 30077333). Tubulin was stained using an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889)(red) at 1/200 dilution. The nuclear counterstain was DAPI (Blue).

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

Immunohistochemical analysis of paraffin-embedded human skin tissue labeling DC-SIGN + DC-SIGNR with ab245115 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Cytoplasmic staining on dendritic cell-like cells of human skin (PMID : 11859097; PMID : 11859097) is observed. Counterstained with hematoxylin.

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

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling DC-SIGN + DC-SIGNR with ab245115 at 1/100 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Cytoplasmic staining on dendritic cell-like cells of human tonsil (PMID : 11859097) is observed. Counterstained with hematoxylin.

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

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

DC-SIGN+DC-SIGNR was immunoprecipitated from 0.35 mg human tonsil tissue lysate using ab245115 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab245115 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1000 dilution.

Lane 1 : Human tonsil tissue lysate 10 μg (input).
Lane 2 : ab245115 IP in Human tonsil tissue.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab245115 in Human tonsil lysate. (-).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (ab245115)

Observed band size: 44 kDa

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• WB

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Western blot - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

Exposure time :

Lane 1 : 3 seconds;

Lane 2 : 92 seconds;

Lane 3 : 37 seconds

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (ab245115) at 1/1000 dilution

Lane 1:

Human lymph node tissue lysate at 20 µg

Lane 2:

Human skin tissue lysate at 20 µg

Lane 3:

Human thymus tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Observed band size: 46 kDa

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• WB

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Western blot - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (AB245115)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-DC-SIGN + DC-SIGNR antibody [EPR22395-72] (ab245115) at 1/1000 dilution

Lane 1:

Recombinant Human DC-SIGN/CD209 Protein (aa59-404) 20 ng

Lane 2:

Recombinant Human DC-SIGNR/CD299 Fc Chimera Protein (aa73-376) 20 ng

Secondary

All lanes:

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

Observed band size: 48 kDa,66 kDa

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Exposure time: 3s