Anti-CISD1/MitoNEET antibody [EPR29116-27]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CISD1 KO HeLa (human cervical adenocarcinoma epithelial cell), ab265276 cells labelling MitoNEET with ab318199 at 1/100 (5.1 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing mitochondrial staining in wildtype HeLa cells (shown in green), and negative staining in CISD1 knockout HeLa cells. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/1000 1ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab318199 at 1/100 (5.1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 2 ug/mL dilution.
-ve control 2 : Anti-COX IV mouse monoclonal 1/1000 1ug/ml dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling MitoNEET with ab318199 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cerebrum. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type Hela (human cervical adenocarcinoma epithelial cell) cell pellet; (B) CISD1 knockout Hela cell pellet tissue labeling MitoNEET with ab318199 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) wild-type Hela cell pellet, no staining on (B) human CISD1 knockout Hela cell pellet. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Human ovarian carcinoma tissue labeling MitoNEET with ab318199 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human ovarian carcinoma. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling MitoNEET with ab318199 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling MitoNEET with ab318199 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat colon. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Mouse liver cancer tissue labeling MitoNEET with ab318199 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse liver cancer. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling MitoNEET with ab318199 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling MitoNEET with ab318199 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse liver. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling MitoNEET with ab318199 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat liver. The section was incubated with ab318199 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling MitoNEET with ab318199 at 1/100 (5.1 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing mitochondrial staining in NIH/3T3 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/1000 1ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab318199 at 1/100 (5.1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 2 ug/mL dilution.
-ve control 2 : Anti-COX IV mouse monoclonal 1/1000 1ug/ml dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

• WB

Supplier Data

Western blot - Anti-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

In Western blot, ab318199 was shown to bind specifically to CISD1. Target of interest was observed at 15 kDa in wild-type Hela cell lysates (lane 1) with no signal observed at this size in CISD1 knockout cell line (lane 2) (lane 2, knockout cell line ab265276 / knockout cell lysate ab258817).

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

Exposure time : Lane 1-2 : 114 seconds, Lane 3-5 : 59 seconds

All lanes:

Western blot - Anti-CISD1/MitoNEET antibody [EPR29116-27] (ab318199) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

CISD1 knockout HeLa whole cell lysate at 20 µg

Lane 3:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 5:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 15 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

All lanes:

Western blot - Anti-CISD1/MitoNEET antibody [EPR29116-27] (ab318199) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Mouse cerebellum tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue lysate at 20 µg

Lane 5:

Rat cerebellum tissue lysate at 20 µg

Lane 6:

Rat liver tissue lysate at 20 µg

Lane 7:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 15 kDa

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-CISD1/MitoNEET antibody [EPR29116-27] (AB318199)

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

In Western blot, Anti-AIF antibody [E20] (ab32516) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-CISD1/MitoNEET antibody [EPR29116-27] (ab318199) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) cytoplasmic fraction at 20 µg

Lane 2:

HeLa mitochondrial fraction at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 15 kDa,36 kDa,67 kDa

false

Exposure time: 10s