Anti-TIM 1 antibody [RM1294]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TIM 1 antibody [RM1294] (AB323414)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Huh7 (human hepatocellular carcinoma epithelial cell) cells labelling TIM 1 with ab323414 at 1/100 (4.94 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 and membrane staining in Huh7 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : 293T (PMID : 23084921)
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.

• Flow Cyt

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Flow Cytometry - Anti-TIM 1 antibody [RM1294] (AB323414)

Flow cytometric analysis of 293T (human embryonic kidney epithelial cell, Left) / Huh7 (human hepatocellular carcinoma epithelial cell, Right) cells labelling TIM 1 with ab323414 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

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

Negative control : 293T.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TIM 1 antibody [RM1294] (AB323414)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A20 (mouse reticulum sarcoma B lymphocyte) cells labelling TIM 1 with ab323414 at 1/100 (4.94 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 membrane staining in A20 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : MEF.
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-TIM 1 antibody [RM1294] (AB323414)

Immunohistochemical analysis of paraffin-embedded (A) Injured mouse kidney (cisplatin-induced acute kidney injury). (B) Control mouse kidney. tissue labeling TIM 1 with ab323414 at 1/2000 (0.247 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on (A) Injured mouse kidney (cisplatin-induced acute kidney injury), no staining on (B) control mouse kidney. The section was incubated with ab323414 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) was used.

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

• Flow Cyt

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Flow Cytometry - Anti-TIM 1 antibody [RM1294] (AB323414)

Flow cytometric analysis of MEF (mouse embryo fibroblast, Left) / A20 (mouse reticulum sarcoma B lymphocyte, Right) cells labelling TIM 1 with ab323414 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Low expression : MEF.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TIM 1 antibody [RM1294] (AB323414)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling TIM 1 with ab323414 at 1/2000 (0.247 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Negative control : no staining on mouse spleen. The section was incubated with ab323414 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) was used.

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-TIM 1 antibody [RM1294] (AB323414)

TIM 1 was immunoprecipitated from 0.35 mg A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate with ab323414 at 1/1000 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323414 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-TIM 1 antibody [RM1294] (ab323414) at 1/1000 dilution

Lane 1:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate at 10 µg

Lane 2:

ab323414 at 1/30 IP in A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate 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 ab323414 in A20 whole cell lysate 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: 70 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-TIM 1 antibody [RM1294] (AB323414)

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

Negative control : 293T (PMID : 23084921, PMID : 12138159), HeLa

The molecular weight observed is consistent with what has been described in the literature (PMID : 12138159, PMID : 35073759).

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

All lanes:

Western blot - Anti-TIM 1 antibody [RM1294] (ab323414) at 1/500 dilution

Lane 1:

Huh7 (human hepatocellular carcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 3:

293T (human embryonic kidney epithelial cell) fresh 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: 100 kDa,36 kDa

false

Exposure time: 48s

• WB

Supplier Data

Western blot - Anti-TIM 1 antibody [RM1294] (AB323414)

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

Low expression : MEF and Neuro-2a

The molecular weight observed is consistent with what has been described in the literature (PMID : 12138159, PMID : 37460623).

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

All lanes:

Western blot - Anti-TIM 1 antibody [RM1294] (ab323414) at 1/1000 dilution

Lane 1:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 3:

Neuro-2a (mouse neuroblastoma neuroblast) 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: 70 kDa,36 kDa

false

Exposure time: 81s

• WB

Supplier Data

Western blot - Anti-TIM 1 antibody [RM1294] (AB323414)

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

Negative control : kidney (PMID : 37460623), spleen

The molecular weight observed is consistent with what has been described in the literature (PMID : 12138159, PMID : 37460623).

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

All lanes:

Western blot - Anti-TIM 1 antibody [RM1294] (ab323414) at 1/1000 dilution

Lane 1:

Mouse injured kidney tissue lysate (cisplatin-induced acute kidney injury) at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse spleen tissue 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: 70 kDa,36 kDa

false

Exposure time: 26s