Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells (Human epithelial cells from cervix adenocarcinoma) labeling Hsp70 with ab182844 at 1/100 dilution, followed by Alexa Fluor®488 Goat Anti-Rabbit IgG H&L (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing both cytoplasmic and nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab182844 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

All lanes : Anti-Hsp70 antibody [EPR17677] (ab182844) at 1/50000 dilution

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate untreated at 37°C
Lane 2 : MCF7 whole cell lysate heat shock (43°C) treated for 1 hour
Lane 3 : MCF7 whole cell lysate heat shock (43°C) treated for 2 hours
Lane 4 : MCF7 whole cell lysate heat shock (43°C) treated for 3 hours
Lane 5 : MCF7 whole cell lysate heat shock (43°C) treated for 4 hours
Lane 6 : MCF7 whole cell lysate heat shock (43°C) treated for 5 hours
Lane 7 : MCF7 whole cell lysate heat shock (43°C) treated for 6 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

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


Exposure time: 30 seconds

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

All lanes : Anti-Hsp70 antibody [EPR17677] (ab182844) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : A431 (Human epidermoid carcinoma) whole cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate

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 [EPR17677] (ab182844) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG 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 breast tissue labeling Hsp70 with ab182844 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and cytoplasmic staining on epithelial cells of Human mammary glands is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Human prostate tissue labeling Hsp70 with ab182844 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and cytoplasmic staining on epithelial cells of Human prostate is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Human lung squamous cell carcinoma tissue labeling Hsp70 with ab182844 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and cytoplasmic staining on cancer cells of Human lung squamous cell carcinoma is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Human transitional cell carcinoma of bladder tissue labeling Hsp70 with ab182844 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and cytoplasmic staining on cancer cells of Human transitional cell carcinoma of bladder is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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 HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Hsp70 with ab182844 at 1/30 dilution (red) compared with a rabbit monoclonal IgG isotype 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.

Hsp70 was immunoprecipitated from HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab182844 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab182844 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: HeLa whole cell extract (Input) 10 µg.

Lane 2: ab182844 IP in HeLa whole cell extract.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab182844 in HeLa whole cell extract.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.