Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Grp75/MOT (green) with ab2799. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were incubated with (left panel) or without (right panel) ab2799 (1:50) for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat-anti-mouse IgG secondary antibody (1:400) for 30 minutes at room temperature. F-Actin (red) was stained with Dylight 554 phalloidin, and nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

Immunohistochemistry was performed on normal biopsies of deparaffinized human testis tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer and microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a Mouse Monoclonal Antibody recognizing Grp75/MOT (ab2799) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunocytochemistry/ Immunofluorescence of P19 (Mouse embryonal carcinoma cell line) cells using ab2799.

Immunocytochemistry/ Immunofluorescence of NS-1 (Mouse myeloma cell line) cells using ab2799.

Immunocytochemistry/ Immunofluorescence of HMVEC (Human microvascular endothelial cell line) cells using ab2799.

Immunocytochemistry/ Immunofluorescence of A549 (Human lung carcinoma cell line) cells using ab2799.

ab2799 (1µg/ml) staining GrP75/MOT in human frontal cerebral cortex using an automated system (DAKO Autostainer Plus). Using this protocol there is strong mitochondrial staining.
Insert depicts negative control (no primary antibody).
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunocytochemistry/Immunofluorescence analysis of human fibroblasts labelling Grp75/MOT with ab2799.