Anti-COL1A2 antibody [RM1295]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COL1A2 antibody [RM1295] (AB322963)

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labeling COL1A2 with ab322963 at 1/10000 (0.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stromal cells of human colon cancer.
The section was incubated with ab322963 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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-COL1A2 antibody [RM1295] (AB322963)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell, Left) / WI-38 (human fetal lung fibroblast, Right) cells labellng COL1A2 with ab322963 at 1/5000 dilution (0.01 ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : HeLa.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-COL1A2 antibody [RM1295] (AB322963)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized WI-38 (human fetal lung fibroblast) cells labelling COL1A2 with ab322963 at 1/100 (5.11 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in WI-38 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : HeLa.
Left Panel is applied with Anti-COL1A2 antibody [RM1295] (ab322963) at 1/100 dilution (5.11 ug/ml);
Right panel is applied with Anti-COL1A2 antibody (ab96723) at 1/100 dilution (13.3 ug/ml).
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 (Magenta). 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 2ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-COL1A2 antibody [RM1295] (AB322963)

Immunohistochemical analysis of paraffin-embedded Human skin tissue labeling COL1A2 with ab322963 at 1/10000 (0.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stromal cells of human skin.
The section was incubated with ab322963 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-COL1A2 antibody [RM1295] (AB322963)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling COL1A2 with ab322963 at 1/10000 (0.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : positive staining on blood vessles of human cerebrum.
The section was incubated with ab322963 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-COL1A2 antibody [RM1295] (AB322963)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling COL1A2 with ab322963 at 1/10000 (0.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on stromal cells of human colon.
The section was incubated with ab322963 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

• IP

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Immunoprecipitation - Anti-COL1A2 antibody [RM1295] (AB322963)

COL1A2 was immunoprecipitated from 0.35 mg WI-38 (human fetal lung fibroblast) whole cell lysate with ab322963 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322963 at 1/1000 dilution.

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible in lanes 4-6.

Lanes 1-3 : 180 seconds, Lanes 4-6 : 58 seconds.

All lanes:

Immunoprecipitation - Anti-COL1A2 antibody [RM1295] (ab322963) at 1/1000 dilution

Lane 1:

(Input) WI-38 (human fetal lung fibroblast) whole cell lysate (Frozen) at 10 µg

Lanes 2 and 5:

ab322963 at 1/30 IP in WI-38 (human fetal lung fibroblast) whole cell lysate (Frozen) at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab322963 in WI-38 whole cell lysate (Frozen) at 10 µg

Lane 4:

(Input) WI-38 (human fetal lung fibroblast) whole cell lysate (Fresh) at 10 µg

Lane 6:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab322963 in WI-38 whole cell lysate (Fresh) at 10 µg

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

Observed band size: 160 kDa

false

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-COL1A2 antibody [RM1295] (AB322963)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MEF (mouse embryo fibroblast) cells labelling COL1A2 with ab322963 at 1/100 (5.11 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in MEF cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : Neuro-2a.
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 (Magenta). 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 2ug/ml dilution.

• WB

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Western blot - Anti-COL1A2 antibody [RM1295] (AB322963)

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

Negative control : HeLa.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23311625, PMID : 24599274 and PMID : 29162919).

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

All lanes:

Western blot - Anti-COL1A2 antibody [RM1295] (ab322963) at 1/1000 dilution

Lane 1:

WI-38 (human fetal lung fibroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

WI-38 transfected with siRNA specifically targeting COL1A2 whole cell lysate at 20 µg

Lane 3:

IMR-90 (human lung fibroblast) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervical adenocarcinoma epithelial cell) 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 1/20000 dilution

Observed band size: 30-160 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-COL1A2 antibody [RM1295] (AB322963)

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

Low expression : brain.

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

All lanes:

Western blot - Anti-COL1A2 antibody [RM1295] (ab322963) at 1/1000 dilution

Lane 1:

Rat skin tissue lysate at 40 µg

Lane 2:

Rat brain tissue lysate at 40 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: 129 kDa,36 kDa

false

Exposure time: 103s