Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Hsp70 with ab181606 at 1/50 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Nuclear and cytoplasm staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (Tubulin mouse mAb) at 1/500 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows;
1. ab181606 at 1/50 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab 150077 (Goat anti rabbit IgG (Alexa Fluor®488) secondary antibody at 1/400 dilution.

 

All lanes : Anti-Hsp70 antibody [EPR16892] (ab181606) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : 293T (Human epithelial cells from embryonic kidney) whole cell lysates
Lane 3 : A431 (Human epidermoid carcinoma) whole cell lysates
Lane 4 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Hsp70 antibody [EPR16892] (ab181606) at 1/1000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Hsp70 antibody [EPR16892] (ab181606) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Mouse spleen lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat heart lysate
Lane 7 : Rat kidney lysate
Lane 8 : Rat spleen lysate
Lane 9 : C6 (Rat glial tumor cells) whole cell lysates
Lane 10 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 12 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human squamous cell carcinoma of cervix tissue labeling Hsp70 with ab181606 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasm staining on tumor cells of Human squamous cell carcinoma of cervix is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Hsp70 with ab181606 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasm staining on epithelial cells of rat testis is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling Hsp70 with ab181606 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining on epithelial cells of mouse testis is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse cerebral cortex tissue labeling Hsp70 with ab181606 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm and Nucleus staining on neuron cells of mouse cerebral cortex tissue is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 2% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling Hsp70 with ab181606 at 1/230 dilution (red) compared with a rabbit monoclonal IgG control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.