Recombinant Anti-Insulin antibody [RM1019] (ab282459)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labelling Insulin with ab282459 at 1/10000 (0.055 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on the islets in human pancreas. The section was incubated with ab282459 overnight at 4°C. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Beta-TC-6 cells labelling Insulin with ab282459 at 1/1000 (0.551 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 cytoplasmic staining in Beta-TC-6 cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Negative control: C2C12 (PMID: 22973301)

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

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

This blot was developed using a higher sensitivity ECL substrate.

 Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling Insulin with ab282459 at 1/10000 (0.055 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on the islet in mouse pancreas. The section was incubated with ab282459 overnight at 4°C. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0).

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

Negative control: C2C12 (PMID: 22973301)

Exposure time: 26 seconds.

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling Insulin with ab282459 at 1/10000 (0.055 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). Positive staining on the islet in rat pancreas. The section was incubated with ab282459 overnight at 4°C. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0).

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling Insulin with ab282459 at 1/10000 (0.055 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer). The section was incubated with ab282459 overnight at 4°C. Counterstained with Hematoxylin.

Negative control: no staining in human skeletal muscle.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse pancreas tissue labeling Insulin with ab282459 at 1/1000 (0.565 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution (Green). Cytoplasmic staining on mouse pancreas islet is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat pancreas tissue labeling Insulin with ab282459 at 1/1000 (0.551 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 ug/ml) dilution (Green). Cytoplasmic staining on rat pancreas islet is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)at 1/1000 (2 ug/ml) dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C2C12 (Mouse myoblasts myoblast) (Left) / Beta-TC-6 (Mouse pancreas insulinoma beta cell) (Right) cells labelling Insulin with ab282459 at 1/5000 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, ab98507) at 1/500 dilution was used as the secondary antibody.

Negative control: C2C12 (PMID: 22973301)

Insulin was immunoprecipitated from 0.35 mg Mouse pancreas lysate 10 ug with ab282459 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab282459 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse pancreas lysate 10 ug

Lane 2: ab282459 IP in Mouse pancreas lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab282459 in Mouse pancreas lysate

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

Exposure time: 3 min.

ELISA using ab282459 at varying antibody concentrations and antigen concentration at 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 was used as the secondary antibody.