Immunohistochemistry analysis of Phospho-IR pTyr1158 showing staining in the cytoplasm of paraffin-embedded human skeletal muscle tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H₂O₂-methanol for 15 min at room temperature, washed with ddH₂O and PBS, and then probed with ab277763 diluted in 3% BSA-PBS at a dilution of 1/20 overnight at +4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prepare for mounting.

Immunofluorescence analysis of Phospho-IR pTyr1158 was performed using 70% confluent log phase HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 10 nM Insulin for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with ab277763 at 2 μg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate a dilution of 1/2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor 555 Rhodamine Phalloidin (1/300 dilution). Panel d represents the merged image showing cytoplasmic localization. Panel e shows untreated cells with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

All lanes : Anti-Insulin Receptor (phospho Y1158) antibody [8HCLC] (ab277763) at 1 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate treated with Insulin (100 ng/mL, 15 min)
Lane 2 : Preincubation with the phosphopeptide, HeLa whole cell lysate treated with Insulin (100 ng/mL, 15 min)

Predicted band size: 156 kDa