Recombinant 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.

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.

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.

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.

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.

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.

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.

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.

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.

Blocking/Dilution buffer: 5% NFDM/TBST.

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

Blocking/Dilution buffer: 5% NFDM/TBST.

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

Blocking/Dilution buffer: 5% NFDM/TBST.

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