Anti-C3 antibody [EPR19394]

• WB

Supplier Data

Western blot - Anti-C3 antibody [EPR19394] (AB200999)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-C3 antibody [EPR19394] (ab200999) at 1/2000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 187 kDa

Observed band size: 115 kDa,187 kDa,68 kDa

false

Exposure time: 30s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of formalin fixed paraffin embedded human liver labelling C3/C3b with ab200999 at a concentration of 0.01 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

Anti-C3/C3b antibody [EPR19394] ab200999 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling C3 with ab200999 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

The plasma in blood vessels of rat kidney was positive staining. The staining pattern is similar to what has been observed in the literature (PMID : 23104558).

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling C3 with ab200999 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on hepatocytes of rat liver is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling C3 with ab200999 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

The plasma in blood vessel of mouse cardiac muscle was positive staining. The staining pattern is similar to what has been observed in the literature (PMID : 23104558).

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling C3 with ab200999 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on hepatocytes of mouse liver is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling C3 with ab200999 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

The plasma in blood vessels of rat lung was positive staining. The staining pattern is similar to what has been observed in the literature (PMID : 23104558).

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling C3 with ab200999 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

The plasma in blood vessels of mouse cerebral cortex was positive staining.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded (A) kidney tissue from wild-type C57BL/6JGpt mice and (B) kidney tissue from C3 knockout mice tissue labeling C3 with ab200999 at 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on (A) kidney tissue from wild-type C57BL/6JGpt mice and no staining on (B) kidney tissue from C3 knockout mice.
The section was incubated with ab200999 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.
The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and C3-KO homozygous mice (Strain ID : T012596).

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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-C3 antibody [EPR19394] (AB200999)

Immunohistochemical analysis of paraffin-embedded (A) Liver tissue from wild-type C57BL/6JGpt mice and (B) Liver tissue from C3 knockout mice tissue labeling C3 with ab200999 at 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on (A) Liver tissue from wild-type C57BL/6JGpt mice and no staining on (B) Liver tissue from C3 knockout mice.
The section was incubated with ab200999 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.
The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and C3-KO homozygous mice (Strain ID : T012596).

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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• WB

Unknown

Western blot - Anti-C3 antibody [EPR19394] (AB200999)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-5 : 8 seconds; Lane 6 : 3 minutes.

All lanes:

Western blot - Anti-C3 antibody [EPR19394] (ab200999) at 1/2000 dilution

Lane 1:

Human serum at 20 µg

Lane 2:

Human plasma at 20 µg

Lane 3:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Mouse plasma at 20 µg

Lane 5:

Rat plasma at 20 µg

Lane 6:

Human milk at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 187 kDa

Observed band size: 115 kDa,187 kDa,43 kDa,68 kDa

false

• WB

Lab

Western blot - Anti-C3 antibody [EPR19394] (AB200999)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

The identity of the lower MW band at approximately 25 kDa (in lane 1-4) is unknown.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and C3-KO homozygous mice (Strain ID : T012596).

All lanes:

Western blot - Anti-C3 antibody [EPR19394] (ab200999) at 1/1000 dilution

Lane 1:

Wild-type mouse kidney tissue lysate (male) at 20 µg

Lane 2:

Wild-type mouse kidney tissue lysate (female) at 20 µg

Lane 3:

C3 knockout mouse kidney tissue lysate (male case1) at 20 µg

Lane 4:

C3 knockout mouse kidney tissue lysate (male case2) at 20 µg

Lane 5:

Wild-type mouse spleen tissue lysate (male) at 20 µg

Lane 6:

Wild-type mouse spleen tissue lysate (female) at 20 µg

Lane 7:

C3 knockout mouse spleen tissue lysate (male case1) at 20 µg

Lane 8:

C3 knockout mouse spleen tissue lysate (male case2) at 20 µg

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 1/20000 dilution

Observed band size: 43-187 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Anti-C3 antibody [EPR19394] (AB200999)

Western blot : Rabbit Monoclonal [EPR19394] to C3/C3b ab200999 staining at 1/2000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 170 kDa in Wild-type 3T3-L1 after treatment with brefeldin A, with no signal observed at this size in C3 knockout 3T3-L1 samples. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-C3 antibody [EPR19394] (ab200999) at 1/2000 dilution

Lane 1:

Wild-type 3T3-L1 Treated: Brefeldin A (300 ng/mL, 24 h) cell lysates at 20 µg

Lane 2:

Wild-type 3T3-L1 Vehicle Control for Brefeldin A (0 ng/mL, 24 h) cell lysates at 20 µg

Lane 3:

C3 knockout 3T3-L1: Treated: Brefeldin A (300 ng/mL, 24 h) cell lysates at 20 µg

Lane 4:

C3 knockout 3T3-L1 Vehicle Control for Brefeldin A (0 ng/mL, 24 h) cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 187 kDa

Observed band size: 170 kDa

false

• WB

Supplier Data

Western blot - Anti-C3 antibody [EPR19394] (AB200999)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1,2,3,5 and 6 : 3 minutes; Lane 4,7 and 8 : 10 seconds.

All lanes:

Western blot - Anti-C3 antibody [EPR19394] (ab200999) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse kidney lysate at 10 µg

Lane 3:

Mouse spleen lysate at 10 µg

Lane 4:

Mouse heart lysate at 10 µg

Lane 5:

Rat brain lysate at 10 µg

Lane 6:

Rat spleen lysate at 10 µg

Lane 7:

Rat heart lysate at 10 µg

Lane 8:

Rat kidney lysate at 10 µg

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

Predicted band size: 187 kDa

Observed band size: 115 kDa,187 kDa,43 kDa,68 kDa

false