Anti-Collagen VI antibody [EPR17072]

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-Collagen VI antibody [EPR17072] (AB182744)

Fluorescence multiplex immunohistochemical analysis of human adrenal gland (formalin-fixed paraffin-embedded section). Panel A shows merged staining of anti-PCSK2 stained on adrenal medulla (ab274418; gray; Opal™690) at 1 : 2000 (0.263 μg/ml) [Panel B], anti-CYP11A1 stained on adrenal cortex (ab272494; green; Opal™520) at 1 : 10000 (0.053 μg/ml) [Panel C], and anti-Collagen VI stained on extracellular matrix (ab182744; red; Opal™570) at 1 : 1000 (1.518 μg/ml) [Panel D] on human adrenal gland. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary antibody. 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. The section was incubated in three rounds of staining : in the order of ab274418, ab272494, and ab182744 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen VI antibody [EPR17072] (AB182744)

Multiplex immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue.

Panel A : Merged staining of Collagen VI (ab182744; green) anti-CD68 (ab213363; red) and anti-Lamin B1 (ab229025; magenta).

Panel B : Anti-Collagen VI (green) stained on extracellular matrix.

Panel C : Anti-CD68 (red) stained on Kupffer cells.

Panel D : Anti-Lamin B1 (magenta) stained on nuclear envelope.

Key protocol steps : The section was incubated in three rounds of staining with ab182744 (1/1000 dilution) ab213363 (1/1000 dilution) and ab229025 (1/4000 dilution) for 30 mins at room temperature. Each round was followed by tyramide signal amplification with the appropriate fluorophore.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

DAPI was used as a nuclear counter stain. A ready-to-use anti-Rabbit and Mouse Polymer HRP was used as a secondary.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen VI antibody [EPR17072] (AB182744)

This data was developed using the same antibody clone in a different buffer formulation (ab271938). Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast labelling Cytokeratin 18 with ab93741 at 1/200 dilution (1.02 µg/mL) (B), Collagen VI with ab271938 at 1/500 dilution (2.084 μg/ml) (C) and Cytokeratin 14 with ab236439 at 1/2000 dilution ( 0.519 μg/ml) (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. Panel A : merged staining of anti-Cytokeratin 14 (magenta; Opal™690), anti-Cytokeratin 18 (green; Opal™520) and anti-Collagen VI (red; Opal™570) on human breast. Panel B : anti-Cytokeratin 18 stained on luminal epithelial cells. Panel C : anti-Collagen VI stained on stroma. Panel D : anti-Cytokeratin 14 stained on myoepithelial cells. The section was incubated in three rounds of staining : in the order of ab236439 for 30 mins, ab93741 for 10 mins, and ab271938 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.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen VI antibody [EPR17072] (AB182744)

Fluorescence multiplex immunohistochemical analysis of human prostate gland tissue (formalin/PFA-fixed paraffin-embedded section). Panel A : merged staining of anti-Collagen VI (ab271938, magenta; Opal™690), anti-Cytokeratin 5 (ab236216, green; Opal™520) and anti-Prostate Specific Antigen (ab76113, red; Opal™570) on human prostate gland tissue. Panel B : anti-Prostate Specific Antigen stained on luminal cells. Panel C : anti-Cytokeratin 5 stained on basal cells. Panel D : anti-Collagen VI stained on stroma. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining : in the order of ab271938 (1/500), ab236216 (1/400), and ab76113 (1/2000) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) was used for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen VI antibody [EPR17072] (AB182744)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat ovary tissue staining AMH with ab272221 at a 1 : 2000 (0.26 μg/ml) dilution, ab182744 Collagen VI used at 1 : 2000 (0.76 ug/ml) dilution and ab270534 DDX4 / MVH used at 1 : 5000 (0.1 μg/ml) dilution.

Panel A : merged staining of anti-AMH (green; Opal™570), anti-Collagen VI (magenta; Opal™690) and anti-DDX4 / MVH (gray; Opal™570) on rat ovary.

Panel B : anti-AMH staining granulosa cells in rat ovary.

Panel C : ant-Collagen VI staining stroma in rat ovary.

Panel D : ant-DDX4 / MVH staining oocytes in rat ovary.

Nuclear DNA was labelled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab272221, ab182744 and ab270534 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.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen VI antibody [EPR17072] (AB182744)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse ovary tissue staining AMH with ab272221 at a 1 : 2000 (0.26 μg/ml) dilution, ab182744 Collagen VI used at 1 : 2000 (0.76 ug/ml) dilution and ab270534 DDX4 / MVH used at 1 : 5000 (0.1 μg/ml) dilution.

Panel A : merged staining of anti-AMH (green; Opal™570), anti-Collagen VI (magenta; Opal™690) and anti-DDX4 / MVH (gray; Opal™570) on mouse ovary.

Panel B : anti-AMH staining granulosa cells in mouse ovary.

Panel C : ant-Collagen VI staining stroma in mouse ovary.

Panel D : ant-DDX4 / MVH staining oocytes in mouse ovary.

Nuclear DNA was labelled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab272221, ab182744 and ab270534 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.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Collagen VI antibody [EPR17072] (AB182744)

This data was developed using ab271938, the same antibody clone in a different buffer formulation.

Immunohistochemistry analysis of Formalin/PFA-fixed paraffin-embedded sections Mouse Prostate tissue labelling E Cadherin with ab324191 at 1 : 500 dilution (B), Cytokeratin 5 with ab236216 at 1 : 1500 dilution (C) and Collagen VI with ab271938 at 1 : 2000 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-E Cadherin (green; Opal™690), anti-Cytokeratin 5 (magenta; Opal™520) and anti-Collagen VI (gray; Opal™570) on mouse prostate.

Panel B : anti-E Cadherin staining epithelium in mouse prostate.

Panel C : ant-Cytokeratin 5 staining basal cells in mouse prostate.

Panel D : ant-Collagen VI staining stoma in mouse prostate.

Nuclear DNA was labelled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab324191, ab236216 and ab271938 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.

• WB

Unknown

Western blot - Anti-Collagen VI antibody [EPR17072] (AB182744)

Lanes 1 - 3 : Merged signal (red and green). Green - ab182744 observed at 109 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab182744 was shown to recognize Collagen VI in wild-type HEK293 cells as signal was lost at the expected MW in COL6A1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and COL6A1 knockout samples were subjected to SDS-PAGE. ab182744 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Collagen VI antibody [EPR17072] (ab182744)

Lane 1:

Wild-type HEK293 whole cell lysate at 20 µg

Lane 2:

COL6A1 knockout HEK293 whole cell lysate at 20 µg

Lane 3:

Human Skeletal Muscle whole cell lysate at 20 µg

Predicted band size: 105 kDa,109 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen VI antibody [EPR17072] (AB182744)

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling Collagen VI with ab182744 at 1/250 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Positive staining on Human cardiac sarcolemma and interstitium is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Heat mediated antigen retrieval was performed 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-Collagen VI antibody [EPR17072] (AB182744)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Collagen VI with ab182744 at 1/250 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Positive staining around basement membranes of Mouse renal tubules is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Heat mediated antigen retrieval was performed 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-Collagen VI antibody [EPR17072] (AB182744)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Collagen VI with ab182744 at 1/250 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Positive staining around sinusoidal endothelial basement membranes is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Collagen VI antibody [EPR17072] (AB182744)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Collagen VI with ab182744 at 1/200 dilution followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows : -

-ve control1 - ab182744 at 1/200 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 - ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Collagen VI antibody [EPR17072] (AB182744)

Immunohistochemical analysis of paraffin-embedded Rat stomach tissue labeling Collagen VI with ab182744 at 1/250 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Positive staining around Rat gastric epithelial basement membranes is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-Collagen VI antibody [EPR17072] (AB182744)

Blocking/Dilution buffer : 5% NFDM/TBST.

Due to posttranslational modifications, observed MW is greater than the predicted MW.

All lanes:

Western blot - Anti-Collagen VI antibody [EPR17072] (ab182744) at 1/2000 dilution

Lane 1:

Mouse heart lysate at 10 µg

Lane 2:

Mouse kidney lysate at 10 µg

Lane 3:

Mouse spleen lysate at 10 µg

Lane 4:

Rat kidney lysate at 10 µg

Lane 5:

Rat spleen lysate at 10 µg

Lane 6:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 109 kDa

Observed band size: 147 kDa

false

• WB

Lab

Western blot - Anti-Collagen VI antibody [EPR17072] (AB182744)

Lanes 1-3 : Merged signal (red and green). Green - ab182744 observed at 136 kDa. Red - loading control ab8245 observed at 36 kDa.

ab182744 Anti-Collagen VI antibody [EPR17072] was shown to specifically react with Collagen VI antibody in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab265060 (knockout cell lysate ab256879) was used. Wild-type and Collagen VI antibody knockout samples were subjected to SDS-PAGE. ab182744 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Collagen VI antibody [EPR17072] (ab182744) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

COL6A1 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human COL6A1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-col6a1-knockout-hek-293t-cell-line-ab265060'>ab265060</a>)

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 109 kDa

Observed band size: 136 kDa

false

• WB

Supplier Data

Western blot - Anti-Collagen VI antibody [EPR17072] (AB182744)

Blocking/Dilution buffer : 5% NFDM/TBST.

Due to posttranslational modifications, observed MW is greater than the predicted MW.

All lanes:

Western blot - Anti-Collagen VI antibody [EPR17072] (ab182744) at 1/20000 dilution

Lane 1:

Human skeletal muscle lysate at 20 µg

Lane 2:

WI-38 (Human fetal lung fibroblast cells) whole cell lysate at 20 µg

Lane 3:

Human placenta lysate at 20 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 109 kDa

Observed band size: 147 kDa

false

• WB

Supplier Data

Western blot - Anti-Collagen VI antibody [EPR17072] (AB182744)

Blocking/Dilution buffer : 5% NFDM/TBST.

Due to posttranslational modifications, observed MW is greater than the predicted MW.

All lanes:

Western blot - Anti-Collagen VI antibody [EPR17072] (ab182744) at 1/2000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 109 kDa

Observed band size: 147 kDa

false